Serum antibody assay for determining protection from malaria, and pre-erythrocytic subunit vaccines

ABSTRACT

Disclosed herein are diagnostic methods and compositions for identifying individuals that are protected against  Plasmodium falciparum  caused malaria. Such methods are particularly useful for determining not only the protective efficacy of Pf whole parasite vaccines for individual subjects, but also within populations of vaccinated subjects. Also disclosed herein are subunit vaccines comprising at least one Pf immunologic determinant for protection against  Plasmodium -caused malaria.

FIELD OF THE INVENTION

This invention relates generally to the field of malaria immunology andvaccinology.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:“2602_0150001_SeqListing_ST25.txt”; Size: 46,696 bytes; and date ofcreation: Mar. 3, 2016) filed herewith the application is incorporatedby reference in its entirety.

BACKGROUND OF THE INVENTION

Malaria control interventions, including insecticide-impregnated bednets, insecticide spraying, and antimalarial drugs, have reduced malariamorbidity and mortality substantially (World Health Organization, WorldMalaria Report: 2013 (2013;www.who/int/malaria/publications/world_malaria_report_2013/report/en/).However, in 2010, despite these measures, there were an estimated 220million clinical cases and 0.66 to 1.24 million deaths caused by malaria(World Health Organization, World Malaria Report: 2012, C. J. Murray etal., Lancet 379, 413-431 (2012)). A highly effective vaccine will beideal for preventing malaria in individuals and eliminating malaria indefined geographic areas. It would optimally target the parasite atasymptomatic, pre-erythrocytic stages (C. V. Plowe et al., J. Infect.Dis. 200, 1646-1649 (2009), malERA Consultative Group on Vaccines, Aresearch agenda for malaria eradication: vaccines. PLoS Med. 8, e1000398(2011)). The World Health Organization malaria vaccine technologyroadmap set a vaccine efficacy goal of 80% by 2025 (Malaria VaccineTechnology Roadmap, 2006;www.malariavaccine.org/files/Malaria_Vaccine_TRM_Final.pdf).

On the other hand, it has been known for over 40 years that high-level,enduring protective immunity can be provided by means of the bitesof >1000 mosquitoes, infected with radiation attenuated Plasmodiumfalciparum (Pf) sporozoites (SPZ) (R. S. Nussenzweig, et al., Nature216, 160-162 (1967); D. F. Clyde, et al., Am. J. Med. Sci. 266, 169-177(1973); K. H. Rieckmann, et al., Trans. R. Soc. Trop. Med. Hyg. 68,258-259 (1974); S. L. Hoffman, et al., J. Infect. Dis. 185, 1155-1164(2002)). However, mosquito bite is not a useful way to administersporozoites and as a practical matter, a whole sporozoite vaccineapproach would require the capacity to manufacture live, aseptic,radiation-attenuated, purified, preserved PfSPZ as the immunogen of aninjectable vaccine that meets regulatory standards (T. C. Luke, et al.,J. Exp. Biol. 206, 3803-3808 (2003); S. L. Hoffman, et al., Hum. Vac. 6,97-106 (2010); J. E. Epstein, et al., Science 334, 475-480 (2011)).

The first clinical trial of PfSPZ Vaccine, comprising the Pf NF54 strainof SPZ (T. Ponnudurai, et al., Trans. R. Soc. Trop. Med. Hyg. 76,242-250 (1982)) was conducted in 80 immunologically naïve adults (J. E.Epstein, et al. (2011)). They received up to 6 doses of 1.35×105 SPZsubcutaneously (SC) or intradermally (ID). PfSPZ Vaccine proved safe andwell-tolerated, but elicited low-level immune response and minimalprotection. It was hypothesized that the limited efficacy was due to theinefficiency of the ID and SC routes of administration (J. E. Epstein,et al. (2011)). Parallel and subsequent studies in non-human primates(NHP) with the PfSPZ Vaccine showed that IV, but not SC, administrationelicited potent and durable PfSPZ-specific T-cell responses inperipheral blood, and most notably in the liver (J. E. Epstein, et al.(2011)), the likely site of immune protection (S. L. Hoffman, et al.,Nat. Med. 6, 1218-1219 (2000)).

Based on these results, a phase 1 clinical trial was conducted todetermine safety, immunogenicity and protective efficacy of IVadministration of PfSPZ Vaccine (R. A. Seder, R. A. et al., Science,341:1359-1365 (2013)—incorporated herein by reference in its entirety).PfSPZ Vaccine-induced protection against Pf malaria was safe, welltolerated and highly protective when administered up to 6 times IV to 40adults. Six of six adult subjects receiving 6.75×10⁵ SPZ in 5 doses wereprotected as were 6 of 9 adult subjects who received 5.4×10⁵ SPZ in 4doses (R. A. Seder, et al. (2013)). Additional clinical trials have nowbeen conducted and are discussed herein.

Malaria vaccine development requires an accurate measure of efficacy.The early signs, symptoms and pathology of malaria are identifiable and,if identified early, malaria can be treated. Controlled human malariainfection (CHMI) of immunized subjects is used to assess protection.While effective, CHMI is cumbersome and expensive, requiringparticipation of several clinicians, experts, and hospital facilities.An assay that is easy to administer and provides high sensitivity(identification of protected individuals) and specificity(identification of unprotected individuals) would therefore be veryuseful.

SUMMARY OF THE INVENTION

Provided herein are methods for determining a state of protectiveimmunity against P. falciparum-induced malaria in a human subject. Insome embodiments, the method comprises testing a human body fluid samplefor antibodies that bind to at least one of a subset of determinants,each of which having a sequence that is at least 85%, 90% 95%, 99% or100% identical to an amino acid sequence comprising a sequence chosenfrom the group consisting of SEQ ID NOs:11-18 or a sequence encoded by acorresponding nucleic acid sequence chosen from the group consisting ofSEQ ID NOs:1-8 or antigenic fragments thereof, and determining whetherthe human body fluid sample comprises antibodies that are specificallyimmunoreactive to the Pf immunologic determinants, whereinimmunoreactivity above a cutoff for at least one recombinant polypeptideindicates a state of protective immunity against P. falciparum-inducedmalaria in the subject.

Further provided are compositions or subunit vaccines comprising atleast one Pf-specific polypeptide comprising an amino acid sequences atleast 85%, 90%, 95%, 99% or 100% identical to an amino acid sequencecomprising a sequence chosen from the group consisting of SEQ IDNOs:11-18 or a sequence encoded by a corresponding nucleic acid sequencechosen from the group consisting of SEQ ID NOs:1-8 or antigenicfragments thereof. Another embodiment is directed to compositions orvaccines comprising nucleic acid sequences chosen from the groupconsisting of SEQ ID NOs:1-8 or antigenic fragments thereof.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B—Antibody reactivity profiles for pre- andpost-immunization with PfSPZ using the high coverage Pf Whole ProteomeMicroarray (representing 91% of the Pf proteome). These heat mapsrepresent the antibody profiles for 1,567 immunoreactive Pf proteins inPfSPZ-immunized volunteers. Rows represent individual antigens. In FIG.1A the columns represent paired samples of preimmunization andpost-immunization/pre CHMI for each subject. In FIG. 1B the columnsrepresent the Delta of pre-immunization and pre-CHMI for each subject.The column headers, shaded in a gradient of light gray to black,represent trial dosing groups as follows (left to right): 1) 4 doses of7.5×10³ PfSPZ; 2) 6 doses of 7.5×10³ PfSPZ; 3) 4 doses of 30×10⁴ PfSPZ;4) 6 doses of 30×10⁴ PfSPZ; 5) 4 doses of 1.35×10⁵ PfSPZ; and 6) 5 dosesof 1.35×10⁵ PfSPZ. In FIG. 1A, pre-immunization and pre-CHMI time pointsare ordered in paired columns, and rows are sorted by mean reactivityacross the study population. The range of responses is depicted in agray scale gradient from light gray to black, as shown in the shadingkey. In FIG. 1B, Deltas for each study subject are shown in a two shadegradient scale, with dark black representing increasing antibodies frombaseline and light gray representing lower antibodies than baseline.

FIG. 2—Immunogenicity profile for 10 antigens of Pf protection by pairedT-test. The comparison of post-immunization vs. pre-immunization wastested using paired T-tests. White bars represent pre-immunization meanantibody levels per antigen, and hashed bars represent post-immunizationantibody levels. Data are stratified by dosing groups. Error barsrepresent the 95% confidence interval of the group means.

FIG. 3—Effect of PfSPZ dose on antibody levels. The volcano plot depictsthe estimated effect of the total dosage (number of PfSPZ administered)on antibody levels. Log change in antibody levels per 10,000 PfSPZreceived is shown on the x-axis with the inverse log P-value on they-axis. The dashed line represents P=0.05, and the triangle pointsrepresent 31 antigens that remained significant after correction for thefalse discovery rate.

FIG. 4—Ten antigens by Wilcoxon rank-sum test between protected andsusceptible volunteers, post immunization/pre-CHMI. The figure presentsa comparison of the median and interquartile ranges of pre-CHMI antibodylevels between protected and unprotected subjects for each antigen. Thevertical “Whisker” bars represent the highest or lowest value within1.5*IQR of the upper and lower quartiles, respectively. The top 10antigens are sorted by P-values from Wilcoxon rank-sum tests. Black dotsrepresent individuals' measurements.

FIG. 5—Deltas for the ten antigens (by Wilcoxon rank-sum test) betweenprotected and susceptible volunteers. The figure presents a comparisonof the median and interquartile ranges of antibody Deltas (pre-CHMIminus pre-immunization) between protected and unprotected subjects foreach antigen. The vertical “Whisker” bars represent the highest orlowest value within 1.5*IQR of the upper and lower quartiles,respectively. The ten antigens are sorted by P-values from Wilcoxonrank-sum tests. Black dots represent individuals' measurements.

FIGS. 6A-6C—Combinatorial analysis of antibodies associated withprotection. The figures represent AUCs modeled on two- tothirty-antibody combinations. The points and lines represent the highestAUCs for linear models fit to varying combinations of antibodies bynumber of features in the model. The triangle line represents meanhighest AUCs for the models fit to all training data sets. The circlelines represent those models fit to the full data set. The diamond linerepresents the AUC of the best fit models in the training data set onthe leave-one-out cross-validation (LOOCV) samples. FIG. 6A representslinear models fit to post-immunization/pre-CHMI antibody levels. FIG. 6Brepresents antibody Deltas between protected and unprotectedindividuals. FIG. 6C represents pre-immunization antibody levels.

FIGS. 7A and 7B—Show the performance of the antibodies in combinatorialmodels of antibody Deltas. The ROC curves represent the true positiverate (sensitivity) and false positive rate (1-specificity) of the linearcombinations of the down-selected list of antibody targets for thecomparison of protected and susceptible vaccines following CHMI. FIG. 7Arepresents a linear model of the top 6 selected antibodies(corresponding to SEQ ID NOs:11-14 & 19-20). FIG. 7B represents thelinear model with the addition of 4 proteins (corresponding to SEQ IDNOs:15-18) to the linear model of FIG. 7A. Solid lines represent ROCcurves modeled on data from all subjects, and dashed lines represent ROCcurves modeled on leave-on-out cross-validation (LOOCV).

FIG. 8—Shows the performance of the 6 Pf antibodies in combinatorialmodels of Peak antibody levels and antibody Deltas and Pre-CHMI antibodylevels and antibody Deltas. The ROC curves represent the true positiverate (sensitivity) and false positive rate (1-specificity) of the linearcombinations of the previously defined list of antibody targets for thecomparison of protected and susceptible vaccines following CHMI(corresponding to SEQ ID NOs:11-14 & 19-20). ROC curves were modeled ondata from all subjects.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “sensitivity” means the degree to which individuals withprotective immunity are identified as positive. In the diagnosticembodiments described herein, sensitivity is the differential formationof complexes comprising an antigen binding molecule, e.g., antibodies inhuman body fluids, and a Pf immunologic determinant (positive reaction)in individuals that are determined to have protective immunity againstmalaria infection.

As used herein, “specificity” means the degree to which individualslacking protective immunity are identified as negative. In thediagnostic embodiments described herein, specificity is the lack ofdifferential formation of complexes comprising an antigen bindingmolecule, e.g., antibodies in human body fluids, and a Pf immunologicdeterminant (negative reaction) in individuals that are determined tolack protective immunity against malaria infection.

As used herein, “controlled human malaria infection,” abbreviated“CHMI”, means a controlled means of infecting individuals with malaria.After vaccination, CHMI serves to challenge subjects by exposing them tomalaria-causing vectors and can be used to distinguish protected andunprotected individuals. CHMI can be accomplished, for example, byexposure of individuals to the bite of mosquitoes infected withPlasmodium or by direct inoculation of Plasmodium sporozoites.

As used herein, “Delta” or “antibody Delta” means the difference betweenthe signals of pre-immunization and post-immunizationimmunoreactivities. In certain embodiments, Deltas are the differencebetween the intensities of measurement of certain Pf immunologicaldeterminants in human body fluid at a time after vaccination minusmeasurement of said Pf immunological determinants in human body fluid ata time before vaccination.

As used herein, “lacking” or to “lack” means being deficient in, or nothaving, a sufficient amount.

As used herein, “immunologic determinant” means an antigen, an antigenicepitope, or a sero-reactive peptide or protein.

As used herein, “differential reactivity” is the difference in theimmunoreactivities of an immunologic determinant with an antigen bindingmolecule, e.g., antibodies in the human body fluids, of protected andunprotected subjects, wherein p-values are less than 0.05 as calculatedusing parametric and non-parametric hypothesis testing methods (e.g.,two-tailed Student's t-test of unequal variance and Wilcoxon's rank-sumtest).

As used herein, an immunologic determinant “encoded by a nucleic acidsequence” means those nucleic acid sequences are transcribed to mRNA,which is translated into the polypeptides which are the immunologicdeterminants.

As used herein, “seroprevalence” means the proportion of a populationwith positive reaction to a given immunoreactivity determination.

As used herein, “probing” a human body fluid means exposing that bodyfluid to one or more immunologic determinants and measuring the specificreactivity of the immunologic determinants to an antigen bindingmolecule, e.g., antibodies, in the human body fluid. As used herein,this measurement is referred to as an “immunoreactivity determination.”

As used herein, a “human body fluid sample” refers to fluid that isremoved from a subject, and can refer to fluid that is unprocessed orfluid that has been processed after removal. In some embodiment, thefluid sample is blood, saliva, excreta, body tissue or tissue fluids. Insome embodiment, the fluid sample is derived from blood, saliva,excreta, body tissue or tissue fluids. In some embodiments, the humanbody fluid sample is serum or plasma.

As used herein, a “detection agent” is a molecule or a combination ofmolecules that specifically recognizes the complex formed by the bindingof an immunologic determinant to an antigen binding molecule (e.g., anantibody).

As used herein, a “solid phase” refers to any structure forimmobilization of immunological determinants, including but not limitedto, glass and plastic (for example, polystyrene or polyvinylidenefluoride) including those treated with protein immobilizing agents suchas poly-lysine, nitrocellulose or porous membranes. Solid phases mayalso include structures in liquid suspension, such as latex or metalmicrobeads, including those treated with protein immobilizing agentssuch as poly-lysine, nitrocellulose or porous membranes.

As used herein, “array” used in conjunction with solid phase refers tothe immobilization of multiple immunological determinants such that eachdeterminant is distinct from the others and a single human body fluidsample may be probed by the solid phase array in a single assay.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides”, and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide or antigenic fragment thereof of the invention may be of asize of about 15 or more, about 20 or more, about 25 or more, about 50or more, about 75 or more, about 100 or more, about 200 or more, about500 or more, about 1,000 or more amino acids. Polypeptides may have adefined three-dimensional structure, although they do not necessarilyhave such structure.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purpose of the invention, as are native orrecombinant polypeptides that have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides and immunogens of the present inventionare fragments, derivatives, analogs, or variants of the foregoingpolypeptides, and any combination thereof. The terms “fragment,”“variant,” “derivative,” and “analog” polypeptides of the presentapplication include any polypeptides that retain at least some of theproperties of the corresponding polypeptide of the application.Fragments of polypeptides of the present invention include proteolyticfragments, as well as deletion fragments, in addition to specificantibody binding fragments discussed elsewhere herein. Variantpolypeptides of the present application include fragments and alsopolypeptides with altered amino acid sequences due to amino acidsubstitutions, deletions, or insertions. Variants may occur naturally orbe non-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions, or additions. Variant polypeptides may also be referred toherein as “polypeptide analogs.” As used herein, a “derivative” of apolypeptide refers to a subject polypeptide having one or more residueschemically derivatized by reaction of a functional side group. Alsoincluded as “derivatives” are those peptides that contain one or morenaturally occurring amino acid derivatives of the twenty standard aminoacids. Derivatives of polypeptides of the present application mayinclude polypeptides that have been altered so as to exhibit additionalfeatures not found on the reference polypeptide of the application.

The term “polynucleotide” is intended to encompass nucleic acids, andrefers to an isolated nucleic acid molecule or construct, e.g.,messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide maycomprise a conventional phosphodiester bond or a non-conventional bond(e.g., an amide bond, such as found in peptide nucleic acids (PNA)). Theterm “nucleic acid” refers to any one or more nucleic acid segments,e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated”nucleic acid or polynucleotide is intended a nucleic acid molecule, DNAor RNA, that has been removed from its native environment. For example,a recombinant polynucleotide encoding polypeptide or antigenic fragmentthereof contained in a vector is considered isolated for the purposes ofthe present invention. Further examples of an isolated polynucleotideinclude recombinant polynucleotides maintained in heterologous hostcells or purified (partially or substantially) polynucleotides insolution. Isolated RNA molecules include in vivo or in vitro RNAtranscripts of polynucleotides of the present invention. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

As used herein, a “coding region” is a portion of nucleic acid thatconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding apolypeptide or antigenic fragment, variant, or derivative thereof.Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA orRNA. In the case of DNA, a polynucleotide comprising a nucleic acid thatencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A “binding molecule” or “antigen binding molecule” of the presentapplication refers in its broadest sense to a molecule that specificallybinds an antigenic determinant of an antigen.

As used herein, “immunoreactive” means the binding of an immunologicdeterminant with an antigen binding molecule. When immunoreactivityoccurs with immunologic determinants of human serum this is alsoreferred to as “sero-reactive”.

In certain embodiments, “specific immunoreactivity” or “specificallyimmunoreact” refers to direct binding to a particular polypeptide or Pfimmunologic determinant.

As used herein, “immunodominant” means an antigenic determinantresponsible in part for the major immune response in a host.

The term “cutoff”, as used herein, refers to a threshold in the antibodylevels, the antibody Delta measurement, or the predictive value. In someembodiments, the immunoreactivity of an immunological determinant abovea certain cutoff is indicative of protective immunity in a subject.Cutoff refers to a single threshold for a single measurement (e.g., aDelta measurement) of a single immunological determinant (e.g., aPf-specific polypeptide).

When combining cutoffs for multiple immunological determinants (e.g., todetermine predictive value) one or more cutoff thresholds may beadjusted plus or minus 20% from the threshold stated for the thresholdof a single determinant to optimize sensitivity and specificity. In someembodiments, the units used to describe the cutoffs herein are antibodyDeltas, which are based on median fluorescence intensity (MFI)measurements from indirect antibody detection and are normalized beforeanalysis (“Normalized MFI”), as described herein. Normalized MFI aretranslatable and scalable to optical density (“OD”) measurements, suchas those measured in ELISA assay. The scale of MFI for the methodsdescribed herein is approximately 0 to 60,000. Typical OD ranges forELISA are from approximately 0.00 to 2.00 or 0.00 to 4.00, depending onthe detection method. Antibody Deltas are translatable and scalable toOD ratios. In some embodiments, cutoffs for antibody Deltas are acquiredby maximizing sensitivity and specificity on the receiver operatingcharacteristic (ROC) curve at varying cutoffs.

The term “predictive value”, as used herein, refers to the predictionestimated from regression models of one or more measurements of theimmunological determinants. In some embodiments, the predictive value isestimated from logistic regression of antibody Delta measurements of oneor more immunological determinants on the probability of being protectedfrom P. falciparum malaria. In some embodiments, the predictive value iscalculated by taking the sum of the products of logistic regressioncoefficients for each immunological determinant and the antibody Deltameasurement for the corresponding immunological determinant (this isperformed for each subject). In certain embodiments, the predictivevalue is used to estimate the performance characteristics of eachcombination of one or more immunological determinants in diagnosingprotection from P. falciparum malaria.

The term “performance characteristics”, as used herein, refers to a setof values estimated to determine the quality of an immunologicaldeterminant for determining if a vaccinated subject is protected from P.falciparum malaria. These estimates include “sensitivity”, “specificity”and “area under the curve (AUC)” for the receiver operatingcharacteristics (ROC) curve for predictive values or cutoff values.

In certain embodiments, as used herein, the term “about” means plus orminus 5% of the numerical value of the number with which it is beingused. Therefore, about 85% means in the range of 80% to 90% as describedherein.

In some embodiments, a binding molecule of the invention is an antibodyor an antigen binding fragment thereof.

In some embodiments, an “antigen binding fragment” refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., Pf antigen). Fragments of a full-length antibody canperform the antigen-binding function of an antibody. Examples of bindingfragments include a Fab fragment, a F(ab′)2 fragment, a Fd fragment; aFv fragment, a single domain antibody fragment or dAb, and scFv.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory Press).

The portion of an antigen or polypeptide that specifically interactswith the antigen binding domain of an antibody is an “epitope,” or an“antigenic determinant.” An antigen or polypeptide may comprise a singleepitope, but typically comprises at least two epitopes, and can includeany number of epitopes, depending on the size, conformation, and type ofantigen.

The terms “antigenic fragment” and “antibody binding fragment” are usedinterchangeably herein. An antigenic fragment, as used herein, is ableto complex with the same antigen binding molecule, e.g., antibody inhuman body fluid, as the immunogenic determinant from which it isderived.

“Conferring protective immunity”, as used herein, refers to providing toa population or a subject (i.e., an individual) the ability to generatean immune response to protect against a disease (e.g., malaria) causedby subsequent exposure to a pathogen (e.g., Plasmodium falciparum) suchthat the clinical manifestations, pathology, or symptoms of disease arereduced during subsequent exposure to the pathogen as compared to anon-treated host, or such that the rate at which infection, or clinicalmanifestations, pathology, or symptoms of disease appear within apopulation are reduced, as compared to a non-treated population.

“Immune response”, as used herein, means a response in the recipient tothe introduction of an immunogen (e.g., attenuated PfSPZ) generallycharacterized by, but not limited to, production of antibodies and/or Tcells. Generally, an immune response may be a cellular response such asinduction or activation of CD4+T cells or CD8+T cells specific forPlasmodium-species epitopes, a humoral response of increased productionof Plasmodium-specific antibodies, or both cellular and humoralresponses. With regard to a malaria vaccine, the immune responseestablished by a vaccine comprising PfSPZ includes but is not limited toresponses to proteins expressed by extracellular sporozoites or otherstages of the parasite after the parasites have entered host cells,especially hepatocytes and mononuclear cells such as dendritic cellsand/or to components of said parasites. In certain embodiments, uponsubsequent challenge by infectious organisms, the immune responseprevents development of pathogenic parasites to the asexual erythrocyticstage that causes disease.

“Immunized”, as used herein, means sufficiently vaccinated to achieve aprotective immune response.

“Indication of Protection,” as used herein, is a statisticallysignificant profile. For example the increase in immunoreactivity to oneor more of an identified subset of Pf-specific antibodies, inindividuals that have been shown to be protected from Pf malaria byother means, for example by challenge using CHMI, and the absence ofsuch profile in unprotected individuals. These Pf-specific antibodiesmay or may not be associated, in whole or in part with the mechanism ofprotection.

“Vaccine”, as used herein, is a preparation comprising an immunogenicagent (also referred to herein as an immunogen) and a pharmaceuticallyacceptable diluent or carrier potentially in combination with excipient,adjuvant and/or additive or protectant. The immunogen may be comprisedof a whole infectious agent or a molecular subset of the infectiousagent (produced by the infectious agent, synthetically orrecombinantly), e.g., a polypeptide, polynucleotide, or fragmentthereof.

In certain embodiments, when the vaccine is administered to a subject,the immunogen stimulates an immune response that will, upon subsequentexposure to an infectious agent, protect the subject from illness ormitigate the pathology, symptoms or clinical manifestations caused bythat agent. In some embodiments, a therapeutic (treatment) vaccine isgiven after infection and is intended to reduce or arrest diseaseprogression. In some embodiments, preventive (prophylactic) vaccine isintended to prevent initial infection or reduce the rate or burden ofthe infection.

In some embodiments, agents used in vaccines against a parasitic diseasesuch as malaria (e.g., P. falciparum vaccine) may be whole-killed(inactive) parasites, live-attenuated parasites (unable to fullyprogress through their life cycle), or purified or artificiallymanufactured molecules associated with the parasite—e.g., recombinantproteins, synthetic peptides, DNA plasmids, and recombinant viruses orbacteria expressing Plasmodium proteins. A vaccine may comprisesporozoites along with other components such as excipient, diluent, orcarrier, for example, human serum albumin. A vaccine may furthercomprise preservative, adjuvant or other immune enhancer, orcombinations thereof, as would be readily understood by those in the art

As used herein, “inoculate” means to administer a clinically relevantdose of a vaccine, or multiple doses over time in a dosage regimen. Insome embodiments, the vaccine is a P. falciparum vaccine, e.g., a P.falciparum whole parasite vaccine.

Diagnostics

In certain embodiments, the current application relates to detection ofparticular combinations of a subset of antibodies that can serve as anindicator of malaria protection, including protective immunity inducedby vaccination, diagnostic assays, identification of subjects in need offurther vaccination, and methods of treatment derived therefrom.

In certain embodiments, the current application relates to measuredchange in the immunoreactivity of a subset of antibodies from an initialstate prior to vaccination to a subsequent state after vaccination. Thedifference serves as an indicator of malaria protection, includingprotective immunity induced by vaccination; and for the identificationof subjects in need of further vaccination.

Certain embodiments of the application are directed to a method fordetermining a state of immunization or protective immunity against P.falciparum-induced malaria in a human subject said method comprisingprobing a human body fluid sample with at least one Pf immunologicdeterminant having an amino acid sequence that is at least 85%, 90%,95%, 99% or 100% identical to a polypeptide with an amino acid sequenceof SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or antigenic fragmentsthereof, or encoded by a polynucleotide with the nucleic acid sequenceof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or antigenic fragments thereof,and determining whether said human body fluid sample comprisesantibodies that are specifically immunoreactive to said Pf immunologicdeterminants, wherein specific immunoreactivity to said Pf immunologicdeterminants indicates protective immunity in said subject. In someembodiments, the method further comprises probing with a further Pfimmunologic determinants having an amino acid sequence that is at least85%, 90%, 95%, 99% or 100% identical to a polypeptide with an amino acidsequence of SEQ ID NO:19, SEQ ID NO:20, or antigenic fragments thereof,or encoded by a polynucleotide with the nucleic acid sequence of SEQ IDNO:9, SEQ ID NO:10, or antigenic fragments thereof.

In some embodiments, the method comprises probing with two or more Pfimmunologic determinants each having an amino acid sequence that is atleast 85%, 90%, 95%, 99% or 100% identical to a polypeptide with anamino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, orantigenic fragments thereof, or encoded by a polynucleotide with thenucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ IDNO:9, or antigenic fragments thereof.

In some embodiments, the method comprises probing with two or more Pfimmunologic determinants each having an amino acid sequence that is atleast 85%, 90%, 95%, 99% or 100% identical to a polypeptide with anamino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO: 19, or SEQ ID NO:20, or antigenic fragments thereof, or encoded by apolynucleotide with the nucleic acid sequence of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, or SEQ ID NO:10, or antigenic fragments thereof.

In some embodiments of the application a human body fluid samplecomprising Pf-specific antibodies is probed with two or more Pfimmunologic determinants each having an amino acid sequence that is atleast 85%, 90%, 95%, 99% or 100% identical to a polypeptide with anamino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:19, or SEQ ID NO:20, or antigenic fragments thereof, orencoded by a polynucleotide with the nucleic acid sequence of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, or SEQ IDNO:10, or antigenic fragments thereof.

In some embodiments, the Pf immunologic determinant comprises DOC2, or acombination of DOC2/MSP5, DOC2/GSK3, DOC2/LRR9, DOC2/MSP5/GSK3,DOC2/MSP5/LRR9, DOC2/UNKPROT/LRR9, DOC2/GSK3/LRR9, DOC2/MSP5/GSK3/LRR9,CSP/DOC2, CSP/DOC2/MSP5, CSP/DOC2/GSK3, CSP/DOC2/LRR9,CSP/DOC2/MSP5/GSK3, or CSP/DOC2/MSP5/LLR9, or antigenic fragmentsthereof.

In some embodiments of the application the human body fluid is serum. Insome embodiments the human body fluid is plasma.

In certain embodiments of the application Pf immunologic determinantsare provided as an array affixed to a solid phase. In certainembodiments antibody immunoreactivity is determined by ELISA. In otherembodiments, immunoreactivity is determined by WESTERN blot analysis. Inother embodiments immunoreactivity is determined by rapid diagnostictests such as lateral flow assays, chromatographic immunoassays, flowthrough nitrocellulose immunoassays and the like, in which antigens areaffixed to a solid phase and contacted by sera, plasma or other humanbody fluid samples. These methods are known in the art. (See, e.g., B.D. Smith, et al., J. Infect. Dis. 204:825-831 (2011)). In otherembodiments immunoreactivity is determined by immunoblot analysis.

In certain embodiments of this application the subject from whom bodyfluid is acquired was previously inoculated with a P.falciparum-specific prophylactic vaccine. In certain embodiments, thedetection of certain Pf-specific antibodies from a subset of the Pfproteome in the body fluids of inoculated individuals is used toidentify immunologic protection against Pf malaria.

Certain embodiments of this application are directed to a compositioncomprising at least one Pf-specific polypeptide each with an amino acidsequence being at least 85%, 90%, 95%, 99% or 100% identical to an aminoacid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or antigenicfragments thereof, or encoded by a polynucleotide having the nucleicacid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or antigenicfragments thereof.

In some embodiments, the composition comprises a further Pf immunologicdeterminant having an amino acid sequence that is at least 85%, 90%,95%, 99% or 100% identical to a polypeptide with an amino acid sequenceof SEQ ID NO:19, SEQ ID NO:20, or antigenic fragments thereof, orencoded by a polynucleotide with the nucleic acid sequence of SEQ IDNO:9, SEQ ID NO:10, or antigenic fragments thereof.

In some embodiments, the composition comprises two or more Pfimmunologic determinants each having an amino acid sequence that is atleast 85%, 90%, 95%, 99% or 100% identical to a polypeptide with anamino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ IDNO: 19, or antigenic fragments thereof, or encoded by a polynucleotidewith the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQID NO:9, or antigenic fragments thereof.

In some embodiments, the composition comprises two or more Pfimmunologic determinants each having an amino acid sequence that is atleast 85%, 90%, 95%, 99% or 100% identical to a polypeptide with anamino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ IDNO:20, or antigenic fragments thereof, or encoded by a polynucleotidewith the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQID NO:10, or antigenic fragments thereof.

In certain embodiments of this application the composition comprises twoor more Pf specific polypeptides each having an amino acid sequence thatis at least 85%, 90%, 95%, 99% or 100% identical to a polypeptide withan amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO: 19, or SEQ ID NO:20, or antigenic fragmentsthereof, or encoded by a polynucleotide with the nucleic acid sequenceof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, orSEQ ID NO:10, or antigenic fragments thereof.

Certain embodiments of this application are directed to methods foridentifying protective immunity against Pf-caused malaria in a humansubject, wherein the protective immunity is identified by the detectionof antibodies in a body fluid sample from said subject that is specificto at least one Pf-specific immunologic determinant affixed to one ormore solid phases, wherein said method comprises:

-   -   a. Applying said human body fluid sample to each solid phase,        wherein each solid phase comprises at least one Pf-specific        polypeptide each having an amino acid sequence that is at least        85%, 90%, 95%, 99% or 100% identical to an amino acid sequence        of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ        ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or        antigenic fragments thereof, or encoded by a polynucleotide        having the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2,        SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,        and SEQ ID NO:8, or antigenic fragments thereof;    -   b. Applying a detection agent that binds antibody-Pf        determinants to the solid phase of (a); and    -   c. Identifying protective immunity against Pf-induced malaria in        said subject by detecting antibody binding to the polypeptides        of (a).

In certain embodiments of this application, identification of protectiveimmunity results from the detection of antibodies in a body fluid samplefrom a human subject that is specific to two or more Pf-specificimmunologic determinants affixed to one or more solid phases, whereinthe method comprises:

-   -   a. Applying said human body fluid sample to each solid phase,        wherein each solid phase comprises at least one Pf-specific        polypeptide each having an amino acid sequence that is at least        85%, 90%, 95%, 99% or 100% identical to an amino acid sequence        of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ        ID NO:19, or SEQ ID NO:20, or antigenic fragments thereof, or        encoded by a polynucleotide having the nucleic acid sequence of        SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9        or SEQ ID NO:10, or antigenic fragments thereof;    -   b Applying a detection agent that binds antibody-Pf determinants        to the solid phase of (a); and    -   d. Identifying protective immunity against Pf-induced malaria in        said subject by detecting antibody binding to the polypeptides        of (a).

Pf Antigens

A subset of ten Pf antigens (SEQ ID NOs:1-10) were identified from thePf proteome based on the immunoreactivity of sera taken afterimmunization as compared to the immunoreactivity of sera taken beforeimmunization in individuals who were protected against CHMI as comparedto those who were not protected. Of these 10, 6 Pf antigens (SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ IDNO:20) were selected for further analysis (Table 9). The LOOCV AUCs forthe combination of these 6 antigens were 0.89 for pre-CHMI antibodylevels and 0.82 for antibody Deltas, while addition of the other 4antigens (SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18)resulted in LOOCV AUCs of 0.91 and 0.81, respectively.

Pf CSP (encoded by SEQ ID NO:9) and MSP5 (encoded by SEQ ID NO:10) weredisclosed in PCT/US2014/053745, filed Sep. 2, 2014, which claimspriority to U.S. 61/872,527, filed Aug. 30, 2013—filed by the sameapplicant as the present application.

GSK3 (SEQ ID NO:14), DOC2 (SEQ ID NO:13), LRR9 (SEQ ID NO:12) andPF3D7_1030200 (SEQ ID NO:10) are disclosed herein.

Pf GSK3 (SEQ ID NO:14) is a 51.6 kDa protein implicated in ATP-bindingand phosphorylation (E. Droucheau, et al., Biochim. Biophys. Acta 1697,181-96 (2004) and has been identified as part of the phosphoproteome (M.Treeck, et al., Cell Host Microbe, 10, 410-9 (2011); E. Lasonder, etal., J. Proteome Res., 11, 5323-37 (2012)). It has been identified as apotential target for drug therapies against Pf malaria (D. I. Osolodkin,et al., Parasitology, 138, 725-35 (2011); F.-J. Gamo, et al., Nature465,305-10 (2010)). Although it has been associated with Maurer's cleftsin the intraerythrocytic cycle (M. Lanzer, et al., Int. J. Parasitol.,36, 23-36 (2006)), a high number of spectra and sequences have beenobserved by mass spectrometry in Pf salivary gland sporozoites (S. E.Lindner, S. E., et al., Mol. Cell. Proteomics 12, 1127-43 (2013)).

Pf DOC2 (SEQ ID NO:13) is a 221 kDa implicated in calcium-dependentphospholipid binding and entry into host cells and has been annotated asa component of cytoplasmic vesicles and host cell plasma membranes. DOC2proteins were shown to be important for microneme secretion and had aneffect on invasion, but not egress, when knocked down (A. Farrell, etal., Science 335, 218-21 (2012)), and it binds calcium when associatedwith membrane fractions (S. Jean, et al., Exp. Parasitol., 144, 91-5(2014)).

Pf LRR9 (SEQ ID NO:12) is a 58.6 kDa with little annotation, hasorthologs in non-human primate species of malaria, but not murinespecies, and has an annotated GO component as integral to membranes.

All three, GSK3, DOC2 and LRR9 have no predicted transmembrane domains,and they may be proteins that induce bystander antibody responses inparallel to alternative mechanism of sterile protection after PfSPZvaccination.

The conserved protein, PF3D7_1030200 (SEQ ID NO:11), is a 51.3 kDaprotein with 4 predicted transmembrane domains, GO component terms forcytoplasm, membrane and nucleus and annotated nucleotide bindingfunction. It also has high levels of expression and multiple spectra bymass spectrometry in sporozoites (Lindner (2013)). Our data suggeststhat this uncharacterized sporozoite protein should be furtherinvestigated for functional immune responses.

The four additional antigens (SEQ ID NOs:15-18) include 3uncharacterized conserved unknown function proteins and a C3HC4-typezinc finger motif (Table 8).

The conserved protein of unknown function, PF3D7_1438800 (SEQ ID NO:5),is an 84 kDa protein with no known transmembrane domains or signalpeptides, but with annotated GO Component Terms for apicoplast,cytoplasm and plasma membranes. Gene expression of said sequence hasbeen detected in the sporozoite and gametocyte stages.

The conserved protein of unknown function, PF3D7_0828100 (SEQ ID NO:16),is a 125 kDa protein with no known transmembrane domains or signalpeptides and no annotated functions. Gene expression of said sequencehas not been detected in the sporozoite stage to date and has beendetected at the gametocyte stage. Our data suggests that thisuncharacterized protein should be further investigated.

The putative C3HC4-type zinc finger protein, PF3D7_0728600 (SEQ IDNO:17), is a 253 kDa protein with no known transmembrane domains orsignal peptides. Its annotated GO Function Terms include protein andzinc ion binding. Said gene sequence shows high gene expression insporozoite and gametocyte stages. Our data suggests that this proteinshould be further investigated.

The conserved protein of unknown function, PF3D7_1468100 (SEQ ID NO:18),is a 296 kDa protein with no known transmembrane domains or signalpeptides, but has a predicted GO Function Term for ATP binding and apredicted GO Component Term for cytoplasm and nucleus. Said protein hasa multitude of spectra by mass spectrometry in sporozoites (Lindner(2013)).

Assay for Identifying Pf Proteins

As disclosed herein, plasma from 32 healthy U.S. volunteers immunizedwith PfSPZ and subjected to controlled human malaria infection (CHMI)was screened using partial and whole Plasmodium falciparum (N) proteomemicroarrays representing 4,805 unique genes, or approximately 91% of thePf proteome. A total of 52 Pf proteins were identified as significantbased on their immunogenic profile, association with sterile protectionafter CHMI and/or they had features associated with protection ingeneralizable combinatorial models.

In certain embodiments, the statistical methods used to determineimmunogenicity (“Immunogenic Profile”): 1) paired T tests of antibodymeasurements before and after vaccination to compare means at each timepoint; 2) ordinary least squares (OLS) regression of PfSPZ dose onantibody Deltas to identify immunogenic dose responses; and 3) linearmixed effects regression (LMER) to model the effect of having beenvaccinated, adjusted by PfSPZ dose, on antibody measurements, addingsubject-level random effects to control for repeated measures.

In certain embodiments, the statistical methods used to determineassociation with sterile protection after CHMI (“Association withProtection”): 1) Estimation of the area under the ROC curve (AUC) forantibody measurements before CHMI and antibody Deltas specific to eachprotein in the classification of subjects as “protected” or“susceptible” as compared to the true diagnosis of protected orsusceptible according to the gold standard of thick and thin blood smearby microscopy (as part of the CHMI protocol); 2) Wilcoxon's rank sumtest of antibody measurements before CHMI and antibody Deltas to compareprotected and susceptible group means.

In certain embodiments, the statistical methods used to determineassociation with protection in generalizable combinatorial models(“Combinatorial Analysis”): 1) fit generalized linear models of antibodymeasurements before vaccination and before CHMI and antibody Deltas onthe probability of being protected (logistic regression) via penalizedmaximum likelihood using lasso and/or elastic net penalization, whilerestricting the models to combinations of a minimum of 2 antibodymeasurements and a maximum of 30 antibody measurements at a time—this isperformed using data from all 32 study subjects; 2) performLeave-One-Out Cross-Validation (LOOCV) by fitting generalized linearmodels of antibody measurements before vaccination and before CHMI andantibody Deltas on the probability of being protected (logisticregression) via penalized maximum likelihood using lasso and/or elasticnet penalization, while restricting the models to combinations of aminimum of 2 antibody measurements and a maximum of 30 antibodymeasurements at a time—this is performed iteratively using data from 31subjects (“Training Set”), varying the subject omitted so that eachsubject is excluded once; 3) Estimate a prediction value for eachsubject for each model generated, and estimate a prediction value foreach “left-out” subject using the corresponding Training Set models; 4)Estimate the AUC of the best model per level of the model fit on alldata (e.g., 2 combination models, 3 combination models . . . 30combination models); 5) Estimate the mean AUC of all Training Set modelsper level of the model (e.g., 2 combination models, 3 combination models. . . 30 combination models); 5) Estimate the AUC of the predictionvalues from the “left-out” subjects per level of the model (e.g., 2combination models, 3 combination models . . . 30 combination models);6) Report the model parameters from the best model fit on all datacorresponding to the level of the models with the highest “left-out”subject AUC.

In addition to the Pf circumsporozoite protein (CSP) and Pf merozoiteprotein 5 (MSP5) (described in PCT/US2014/053745), Pf glycogen synthasekinase 3 (GSK3), Pf double C2-like domain-containing protein (DOC2), Pfleucine-rich repeat protein (LRR9) and a conserved Pf protein of unknownfunction (PF3D7_1030200) were identified, both individually and incombination with each other and with CSP and/or MSP5, as effective inmethods to demonstrate protection against Pf-induced malaria and assubunit vaccine targets.

Four additional proteins (conserved proteins of unknown function(PF3D7_1438800, PF3D7_0828100, and PF3D7_1468100) and zinc finger, C3HC4type (PF3D7_0728600) were also identified.

Controlled Human Malaria Infection (CHMI)

The evaluation of the efficacy of malaria vaccines and treatmentsbenefits from carefully monitoring results in early diagnosis of theonset of the signs, symptoms and pathology of malaria and its rapid andeffective treatment thereafter. CHMI has become a method by which theefficacy of vaccination against malaria can be measured. In the trialdescribed in Seder et al., Science, 341:1359-1365 (2013), CHMI wasachieved by means of exposing subjects to Pf-infected mosquitoes thatmet standard infectivity criteria (K. E. Kester, et al., (2009); S. L.Hoffman, et al. (2002); J. D. Chulay, et al. Am. J. Trop. Med. Hyg. 35,66-68 (1986); L. S. Rickman, et al. Am. J. Trop. Med. Hyg. 43, 441-445(1990); K. E. Lyke, et al., PLoS ONE 5, e13490 (2010)) approximately 3weeks after the last dose of PfSPZ Vaccine. The mosquitoes used wereinfected with Pf NF54 or a clone thereof. Subjects were monitored asoutpatients for 7 days after CHMI and then admitted to the NIH ClinicalCenter for up to 11 consecutive nights or until diagnosis and cure ofparasitemia was documented. Daily thick blood smears were performed,with additional smears performed when needed, based on clinicalpresentation. Treatment with chloroquine or atovaquone/proguanil wasinitiated when a thick blood smear had >2 Pf parasites in 0.5 μL blood,confirmed by an expert malaria microscopist. Subjects were dischargedafter two consecutive days with negative malaria smears or on post-CHMIday 18 if not parasitemic. Subjects were considered protected if smearswere negative through day 28 post-CHMI. Quantitative PCR (qPCR) wasperformed to detect parasite DNA in blood.

In the clinical trial of Seder et al., antibody titers to CSP weremeasured. Two weeks after the final PfSPZ Vaccine administration, therewas a relationship between the total dosage of vaccine and results ofPfCSP ELISA. When used alone, measurement of serum antibodies to CSPexhibits 77% specificity with regard to distinguishing between protectedand unprotected subjects, which is too low to be useful as a clinicalalternative for CHMI. As disclosed herein, the multiplex reactivity ofserum antibodies from human subjects directed to Pf antigens of CSP andMSP5 demonstrate better combined sensitivity and specificity.Furthermore, several of the newly identified subset of antigens, eitheralone or in combination show both high sensitivity and specificity(Table 1) making them an improved alternative to CHMI.

Cutoffs and Predictive Values

In certain embodiments, the predictive values for each study subject areestimated by logistic regression models of antibody Deltas for one ormore immunological determinants on the probability of being protectedagainst P. falciparum CHMI. In some embodiments, the predictive valuesare used to estimate the performance characteristics, also known assensitivity analysis, for a given immunological determinant or acombination of immunological determinants. The resulting sensitivity,specificity and AUC calculations give an indication of the quality ofsaid immunological determinants or combinations thereof. This can beinterpreted that there exists an antibody Deltas cutoff for eachimmunological determinant that can be applied diagnose protection invaccines with the same sensitivity and specificity. Cutoff values forindividual immunological determinants can be estimated (e.g., as shownin Table 1). Cutoff values for multiple immunological determinant testscan vary according to a trade-off between the antibody Deltas for eachimmunological determinant tested. For example, increases in the antibodyDeltas for Immunological Determinant A above the correspondingsingle-immunological determinant cutoff may reduce the required antibodyDeltas level of Immunological Determinant B to a level below thecorresponding single-immunological determinant cutoff. The range ofacceptable cutoff values for a multi-immunological determinant test isdetermined by empirical testing.

In certain embodiments, the cutoff value for each immunologicaldeterminant is used as criteria for diagnosing the state of “protected”or “susceptible” for each subject. Antibody levels are measured asnormalized median fluorescence intensity, or “normalized MFI” (oroptical density [“OD”] or like measurements), for pre-immunizationlevels and post-immunization levels using human bodily fluids takenbefore and after immunization. Then antibody Deltas will be calculatedby subtracting the pre-immunization normalized MFI values (or OD valuesor like) from the post-immunization normalized MFI values (or OD valuesor like). The cutoff values are used to score a subject as “protected”if the antibody Delta(s) exceed the cutoff values or “susceptible” ifthe antibody Delta(s) are below the cutoff values. In certainembodiments, when multiple cutoffs are used in combination as a multipleimmunological determinant test, cutoff values may deviate from thestated optimal cutoff for a single immunological determinant by theempirically determined optimal cutoff for a given combination, deviatingnot more than 20% above or below the stated optimal cutoff, (e.g., about1-20% above, about 5-20% above, about 10-20% above, about 15-20% above,about 1-15% above, about 1-10% above, or about 1-5% above).

TABLE 1 Optimal cutoff values for antibody Deltas (difference betweenpre-immunization and post-immunization immunoreactivities) againstindividual Pf antigens in discriminating protection and susceptibilityto P. falciparum CHMI. Antigens Cutoff * CSP (PF3D7_0304600) 4.90 MSP5(PF3D7_0206900.1) 0.60 Conserved/unknown protein (PF3D7_1030200)** 0.67GSK3 (PF3D7_0312400) 0.68 DOC2 (PF3D7_1243900) 1.36 LRR9 (PF3D7_0906700)0.92 * Cutoff: the cutoff value is the antibody Deltas measurementestimated by maximizing sensitivity and specificity for determiningprotection against P. falciparum CHMI. **As used herein, theconserved/unknown protein PF3D7_1030200 is referred to as“Conserved/unknown protein” or “UNKPROT”.

TABLE 2 Performance characteristics of antibody Deltas (differencebetween pre-immunization and post-immunization immunoreactivities)against combinations of 6 Pf antigens in discriminating protection andsusceptibility to CHMI. Predictive False False Antigens Value *Sensitivity Specificity Positives Negatives CSP (PF3D7_0304600) 4.90 92%79% 4 1 MSP5 (PF3D7_0206900.1) 0.60 85% 79% 4 2 Conserved/unknownprotein (PF3D7_1030200)** 0.67 92% 89% 2 1 GSK3 (PF3D7_0312400) 0.68 85%89% 2 2 DOC2 (PF3D7_1243900) 1.36 92% 95% 1 1 LRR9 (PF3D7_0906700) 0.9285% 84% 3 2 CSP, MSP5 5.13 85% 89% 2 2 CSP, Conserved/unknown protein4.22 92% 84% 3 1 CSP, GSK3 3.52 85% 79% 4 2 CSP, DOC2 4.83 100%  89% 2 0CSP, LRR9 4.04 85% 84% 3 2 MSP5, Conserved/unknown protein 1.79 85% 89%2 2 MSP5, GSK3 1.30 85% 89% 2 2 MSP5, DOC2 1.38 92% 95% 1 1 MSP5, LRR91.30 85% 89% 2 2 Conserved/unknown protein, GSK3 0.98 92% 89% 2 1Conserved/unknown protein, DOC2 2.27 92% 89% 2 1 Conserved/unknownprotein, LRR9 1.59 85% 89% 2 2 GSK3, DOC2 1.68 92% 95% 1 1 GSK3, LRR91.23 85% 89% 2 2 DOC2, LRR9 1.93 92% 95% 1 1 CSP, MSP5,Conserved/unknown protein 4.83 85% 89% 2 2 CSP, MSP5, GSK3 4.32 85% 89%2 2 CSP, MSP5, DOC2 4.83 100%  89% 2 0 CSP, MSP5, LRR9 4.49 85% 89% 2 2CSP, Conserved/unknown protein, GSK3 3.47 85% 84% 3 2 CSP,Conserved/unknown protein, DOC2 5.99 85% 89% 2 2 CSP, Conserved/unknownprotein, LRR9 4.03 85% 84% 3 2 CSP, GSK3, DOC2 4.86 100%  89% 2 0 CSP,GSK3, LRR9 4.13 92% 84% 3 1 CSP, DOC2, LRR9 4.25 100%  89% 2 0 MSP5,Conserved/unknown protein, GSK3 1.77 85% 89% 2 2 MSP5, Conserved/unknownprotein, DOC2 2.25 92% 89% 2 1 MSP5, Conserved/unknown protein, LRR92.52 85% 89% 2 2 MSP5, GSK3, DOC2 1.66 92% 95% 1 1 MSP5, GSK3, LRR9 1.2485% 89% 2 2 MSP5, DOC2, LRR9 1.94 92% 95% 1 1 Conserved/unknown protein,GSK3, DOC2 2.29 92% 89% 2 1 Conserved/unknown protein, GSK3, LRR9 1.1092% 84% 3 1 Conserved/unknown protein, DOC2, LRR9 2.33 92% 95% 1 1 GSK3,DOC2, LRR9 1.94 92% 95% 1 1 CSP, MSP5, Conserved/unknown protein, GSK34.52 85% 89% 2 2 CSP, MSP5, Conserved/unknown protein, DOC2 5.91 85% 89%2 2 CSP, MSP5, Conserved/unknown protein, LRR9 4.35 85% 89% 2 2 CSP,MSP5, GSK3, DOC2 4.86 100%  89% 2 0 CSP, MSP5, GSK3, LRR9 4.71 92% 89% 21 CSP, MSP5, DOC2, LRR9 4.34 100%  89% 2 0 CSP, Conserved/unknownprotein, GSK3, DOC2 6.25 85% 89% 2 2 CSP, Conserved/unknown protein,GSK3, LRR9 4.09 92% 84% 3 1 CSP, Conserved/unknown protein, DOC2, LRR95.38 85% 89% 2 2 CSP, GSK3, DOC2, LRR9 5.78 92% 89% 2 1 MSP5,Conserved/unknown protein, GSK3, DOC2 2.27 92% 89% 2 1 MSP5,Conserved/unknown protein, GSK3, LRR9 2.35 85% 89% 2 2 MSP5,Conserved/unknown protein, DOC2, LRR9 2.29 92% 89% 2 1 MSP5, GSK3, DOC2,LRR9 1.88 92% 95% 1 1 Conserved/unknown protein, GSK3, DOC2, LRR9 2.3392% 89% 2 1 CSP, MSP5, Conserved/unknown protein, GSK3, DOC2 6.21 85%89% 2 2 CSP, MSP5, Conserved/unknown protein, GSK3, LRR9 5.32 85% 89% 22 CSP, MSP5, Conserved/unknown protein, DOC2, LRR9 5.39 85% 89% 2 2 CSP,MSP5, GSK3, DOC2, LRR9 5.75 92% 89% 2 1 CSP, Conserved/unknown protein,GSK3, DOC2, LRR9 5.66 92% 89% 2 1 MSP5, Conserved/unknown protein, GSK3,DOC2, LRR9 2.28 92% 89% 2 1 CSP, MSP5, Conserved/unknown protein, GSK3,DOC2, LRR9 5.60 92% 89% 2 1 * Predictive Value: The predictive valuedisplayed is the optimal logistic regression prediction (“predictivevalue”) for maximizing sensitivity and specificity performancecharacteristics in determining protection against P. falciparum malaria.**As used herein, the conserved/unknown protein PF3D7_1030200 isreferred to as “Conserved/unknown protein” or “UNKPROT”.

As shown in Table 2, 9 combinations (DOC2, DOC2/MSP5, DOC2/GSK3,DOC2/LRR9, DOC2/MSP5/GSK3, DOC2/MSP5/LRR9, DOC2/UNKPROT/LRR9,DOC2/GSK3/LRR9, DOC2/MSP5/GSK3/LRR9) demonstrate 92% sensitivitycombined with 95% specificity and having no more than 1 false positiveand 1 false negative out of the 32 individuals assayed by CHMI. Sixcombinations (CSP/DOC2, CSP/DOC2/MSP5, CSP/DOC2/GSK3, CSP/DOC2/LRR9,CSP/DOC2/MSP5/GSK3, CSP/DOC2/MSP5/LLR9) demonstrate 100% sensitivity and89% specificity and having 2 false positives and no false negatives outof the 32 individuals assayed by CHMI. In total, 15 combinations have nomore than 2 combined false positives and negatives of the 32 individualsassayed.

In certain embodiments, new patients receiving vaccination have bodilyfluid specimens taken before initial vaccination and after completion ofa full vaccine regimen. A diagnostic test is then performed (e.g.,ELISA-, Western- or alternative immunoblotting technique-based)generating pre-immunization and post-immunization antibody levels foreach immunological determinant. The pre-immunization antibody levels aresubtracted from the corresponding post-immunization levels to calculateantibody Deltas for each immunological determinant. Antibody Deltavalues are inserted into the corresponding formula for estimatingpredictive value (as shown in the Table 3). If a subject's predictivevalue exceeds that of the established cutoff criteria (Shown in Table2), the subject is diagnosed as protected from P. falciparum malaria. Ifa subject's predictive value does not exceed the established cutoffcriteria, then the patient is not diagnosed as protected or is diagnosedas not protected. In some embodiments, if the patient is not diagnosedas protected or is diagnosed as not protected, a further vaccinationdose or other anti-malarial intervention can be administered.

TABLE 3 Predictive Value Formulas Antigen Combination Predictive ValueFormula** CSP 1.35 * CSP MSP5 3.48 * MSP5 UNKPROT 2.3 * UNKPROT GSK32.25 * GSK3 DOC2 5.46 * DOC2 LRR9 3.99 * LRR9 CSP, MSP5 1.18 * CSP +2.06 * MSP5 CSP, UNKPROT 1.04 * CSP + 0.718 * UNKPROT CSP, GSK3 0.957 *CSP + 0.873 * GSK3 CSP, DOC2 1.28 * CSP + 4.48 * DOC2 CSP, LRR9 0.951 *CSP + 3.23 * LRR9 MSP5, UNKPROT 2.92 * MSP5 + 2.13 * UNKPROT MSP5, GSK31.34 * MSP5 + 2.04 * GSK3 MSP5, DOC2 0.367 * MSP5 + 5.27 * DOC2 MSP5,LRR9 2.1 * MSP5 + 2.98 * LRR9 UNKPROT, GSK3 1.22 * UNKPROT + 1.56 * GSK3UNKPROT, DOC2 1.51 * UNKPROT + 4.72 * DOC2 UNKPROT, LRR9 1.36 *UNKPROT + 3.05 * LRR9 GSK3, DOC2 1.19 * GSK3 + 3.86 * DOC2 GSK3, LRR91.27 * GSK3 + 2.38 * LRR9 DOC2, LRR9 4.04 * DOC2 + 2.19 * LRR9 CSP,MSP5, UNKPROT 0.868 * CSP + 2.44 * MSP5 + 0.876 * UNKPROT CSP, MSP5,GSK3 0.986 * CSP + 1.71 * MSP5 + 0.576 * GSK3 CSP, MSP5, DOC2 1.28 *CSP + −0.0307 * MSP5 + 4.49 * DOC2 CSP, MSP5, LRR9 0.927 * CSP + 1.7 *MSP5 + 2.31 * LRR9 CSP, UNKPROT, GSK3 0.856 * CSP + 0.432 * UNKPROT +0.758 * GSK3 CSP, UNKPROT, DOC2 1.11 * CSP + 0.503 * UNKPROT * 4.45 *DOC2 CSP, UNKPROT, LRR9 0.933 * CSP + 0.0793 * UNKPROT + 3.2 * LRR9 CSP,GSK3, DOC2 1.3 * CSP + −0.0427 * GSK3 * 4.52 * DOC2 CSP, GSK3, LRR91.02 * CSP + −0.298 * GSK3 + 3.63 * LRR9 CSP, DOC2, LRR9 1.03 * CSP +3.67 * DOC2 + 1.77 * LRR9 MSP5, UNKPROT, GSK3 2.43 * MSP5 + 1.56 *UNKPROT + 0.957 * GSK3 MSP5, UNKPROT, DOC2 1.08 * MSP5 + 1.6 * UNKPROT +4.22 * DOC2 MSP5, UNKPROT, LRR9 2.59 * MSP5 + 1.49 * UNKPROT + 1.74 *LRR9 MSP5, GSK3, DOC2 −0.0717 * MSP5 + 1.19 * GSK3 + 3.88 * DOC2 MSP5,GSK3, LRR9 1.28 * MSP5 + 1 * GSK3 + 2.09 * LRR9 MSP5, DOC2, LRR9−0.0995 * MSP5 + 4.07 * DOC2 + 2.22 * LRR9 UNKPROT, GSK3, DOC2 1.34 *UNKPROT + 0.443 * GSK3 + 4.28 * DOC2 UNKPROT, GSK3, LRR9 1.09 *UNKPROT + 0.879 * GSK3 + 2.17 * LRR9 UNKPROT, DOC2, LRR9 1.24 *UNKPROT + 4.04 * DOC2 + 1.73 * LRR9 GSK3, DOC2, LRR9 0.394 * GSK3 +3.79 * DOC2 + 1.82 * LRR9 CSP, MSP5, UNKPROT, GSK3 0.821 * CSP + 2.27 *MSP5 + 0.776 * UNKPROT + 0.281 * GSK3 CSP, MSP5, UNKPROT, DOC2 1.09 *CSP + 0.27 * MSP5 + 0.525 * UNKPROT + 4.34 * DOC2 CSP, MSP5, UNKPROT,LRR9 0.857 * CSP + 1.92 * MSP5 + 0.311 * UNKPROT + 2.09 * LRR9 CSP,MSP5, GSK3, DOC2 1.3 * CSP + 0.00652 * MSP5 + −0.0434 * GSK3 + 4.52 *DOC2 CSP, MSP5, GSK3, LRR9 1.13 * CSP + 2.54 * MSP5 + −0.919 * GSK3 +3.08 * LRR9 CSP, MSP5, DOC2, LRR9 1.03 * CSP + −0.302 * MSP5 + 3.75 *DOC2 + 1.89 * LRR9 CSP, UNKPROT, GSK3, DOC2 1.16 * CSP + 0.549 *UNKPROT + −0.194 * GSK3 + 4.61 * DOC2 CSP, UNKPROT, GSK3, LRR9 1 * CSP +0.0754 * UNKPROT + −0.297 * GSK3 + 3.61 * LRR9 CSP, UNKPROT, DOC2, LRR90.968 * CSP + 0.309 * UNKPROT + 3.75 * DOC2 + 1.62 * LRR9 CSP, GSK3,DOC2, LRR9 1.16 * CSP + −0.825 * GSK3 + 3.79 * DOC2 + 3.24 * LRR9 MSP5,UNKPROT, GSK3, DOC2 0.91 * MSP5 + 1.45 * UNKPROT + 0.335 * GSK3 + 3.98 *DOC2 MSP5, UNKPROT, GSK3, LRR9 2.38 * MSP5 + 1.41 * UNKPROT + 0.311 *GSK3 + 1.52 * LRR9 MSP5, UNKPROT, DOC2, LRR9 0.703 * MSP5 + 1.3 *UNKPROT + 3.82 * DOC2 + 1.41 * LRR9 MSP5, GSK3, DOC2, LRR9 −0.245 *MSP5 + 0.409 * GSK3 + 3.86 * DOC2 + 1.89 * LRR9 UNKPROT, GSK3, DOC2,LRR9 1.26 * UNKPROT + −0.0668 * GSK3 + 4.08 * DOC2 + 1.81 * LRR9 CSP,MSP5, UNKPROT, 1.15 * CSP + 0.525 * MSP5 + 0.61 * UNKPROT + −0.266 *GSK3 + GSK3, DOC2 4.46 * DOC2 CSP, MSP5, UNKPROT, 1.05 * CSP + 2.83 *MSP5 + 0.393 * UNKPROT + −0.982 * GSK3 + GSK3, LRR9 2.88 * LRR9 CSP,MSP5, UNKPROT, 0.97 * CSP + −0.0678 * MSP5 + 0.303 * UNKPROT + 3.76 *DOC2 + DOC2, LRR9 1.65 * LRR9 CSP, MSP5, GSK3, DOC2, 1.17 * CSP +0.649 * MSP5 + −0.908 * GSK3 + 3.65 * DOC2 + 3.03 * LRR9 LRR9 CSP,UNKPROT, GSK3, 1.09 * CSP + 0.342 * UNKPROT + −0.856 * GSK3 + 3.88 *DOC2 + DOC2, LRR9 3.17 * LRR9 MSP5, UNKPROT, GSK3, 0.775 * MSP5 + 1.34 *UNKPROT + −0.141 * GSK3 + 3.88 * DOC2 + DOC2, LRR9 1.52 * LRR9 CSP,MSP5, UNKPROT, 1.1 * CSP + 1.03 * MSP5 + 0.424 * UNKPROT + −0.99 *GSK3 + 3.67 GSK3, DOC2, LRR9 * DOC2 + 2.8 * LRR9 * Indicates multipliedby the antibody Delta of the corresponding determinant. **In someembodiments, coefficient values in Table 3 can vary up to about 20%,about 15%, about 10%, about 5%, about 2%, about 1%, or about 0.5%.

In certain embodiments, assays have been developed in whichimmunological determinants described herein are affixed in a solidphase, for example, on a chip as a microarray as described herein uponwhich human body fluids are probed and specific antibodies to saidimmunological determinants are detected. In some embodiments these chipsare provided to the clinician for use in assays of the sera fromimmunized individuals to look for reactivity patterns (specifiedcombinations of immunodeterminants) indicating protective efficacy, forexample as part of a clinical trial protocol, either in lieu of or inaddition to CHMI. Additionally, these assays can be used to screen apopulation of subjects that have potentially been exposed to PfSPZ todetermine those in need of vaccination as evidenced by their lacking thereactivity patterns (specified combinations of immunodeterminants)indicating protective efficacy. Probing of the sera of subjects isconducted and evaluated as described above. Alternatively, otherimmunodeterminative tests can be employed to determine sero-reactivityto specific, identified multiplex Pf immunological determinants. Seracan be assessed for antibodies against PfSPZ by immunofluorescence assay(IFA) (J. E. Epstein, et al. (2011)) or to recombinant polypeptideshaving the sequence of SEQ ID NO:11-20 or encoded by SEQ ID NO:1-10 orantigenic fragments thereof or any Pf multiplex combinations thereof byenzyme linked immunosorbent assay (ELISA) as described (R. A. Seder, etal. (2013)). Alternatively, said immunological determinants may be fixedto solid phase membranes, for example, nitrocellulose as performed instandard Western blot, and immunoreactivity of the sera of subjects maybe tested using lateral flow assays that detect antibodies bound toimmunological determinants by protein A-celluloid gold complex toproduce a visible diagnostic test line, as have been developed fordiagnosis of HIV (or example, HIV 1/2 STAT-PAK® tests by ChemBioDiagnostic Systems, Inc., Medford, N.Y.) or Hepatitis C (for example,OraQuick® HCV test by OraSure Technologies, Inc., Bethlehem, Pa.).

Assays of humoral antibodies of vaccinated individuals that are easy toadminister and have substantial sensitivity and specificity foridentifying those individuals that are or are not protected will havegreat value, not only during the clinical testing of malaria vaccines,but also as a public health tool during post licensure vaccinationcampaigns. In addition, assays of the application that can be processedquickly and provide rapid results provide an advantage over currentassays (e.g., CHMI) for protective immunity. The assays disclosed hereinare easy to administer and provide high sensitivity (identification ofprotected individuals) and specificity (identification of unprotectedindividuals).

In some embodiments, the methods of the application are used todetermine a satisfactory end point for vaccination. For example, themethods of the application can be used to determine whether immunizationor protective immunity has been achieved and, optionally, whetherfurther treatment is needed.

In one embodiment, the end point has been achieved and no furthervaccine is administered to the subject when antibodies that specificallyimmunoreact with the specified combinations of immunodeterminantsdisclosed herein.

In another embodiment, the end point is not achieved and a subject isgiven a further dose of, or inoculated with a P. falciparum vaccine whenantibodies that specifically immunoreact the specified combinations ofimmunodeterminants disclosed herein are not detected.

Certain embodiments are directed to a method for determining a state ofprotective immunity against P. falciparum-induced malaria in a humansubject, comprising: (a) probing a human body fluid sample with at leastone recombinant polypeptide comprising a sequence that is 85%, 90%, 95%,99% or 100% identical to an amino acid sequence of SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, or SEQ ID NO:18, or antigenic fragments thereof, or encoded by apolynucleotide having a nucleic acid sequence chosen from SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, or SEQ ID NO:8, or an antigenic fragment thereof; (b) determiningwhether said human body fluid sample comprises an antibody that isspecifically immunoreactive to said at least one recombinantpolypeptide, wherein positive specific immunoreactivity indicates astate of protective immunity in said subject.

Certain embodiments are directed to a method to assess the response to amalaria vaccine in a human subject comprising: (a) testing for thepresence of an antibody binding to at least one recombinant polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100%identical to an amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQID NO:18, or antigenic fragments thereof, or encoded by a polynucleotidehaving a nucleic acid sequence chosen from SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ IDNO:8, or an antigenic fragment thereof in a body fluid sample taken fromthe subject after a first dose of a malaria vaccine is administered tothe subject; and (b) determining that the subject has protectiveimmunity against P. falciparum-induced malaria if the presence ofantibody binding to the at least one recombinant polypeptide from step(a) is detected.

Certain embodiments are directed to a method for determining a state ofprotective immunity against P. falciparum-induced malaria in a humansubject, comprising: (a) probing a human body fluid sample with apolypeptide comprising DOC2 or an antigenic fragment thereof or DOC2 oran antigenic fragment thereof in combination with one of the following:(i) MSP5 or an antigenic fragment thereof, (ii) GSK3 or an antigenicfragment thereof, (iii) LRR9 or an antigenic fragment thereof, (iv) MSP5or an antigenic fragment thereof and GSK3 or an antigenic fragmentthereof, (v) MSP5 or an antigenic fragment thereof, LRR9 or an antigenicfragment thereof, (vi) UNKPROT or an antigenic fragment thereof and LRR9or an antigenic fragment thereof, (vii) GSK3 or an antigenic fragmentthereof and LRR9 or an antigenic fragment thereof, (viii) MSP5 or anantigenic fragment thereof, GSK3 or an antigenic fragment thereof, andLRR9 or an antigenic fragment thereof, (ix) CSP or an antigenic fragmentthereof, (x) CSP or an antigenic fragment thereof and MSP5 or anantigenic fragment thereof, (xi) CSP or an antigenic fragment thereofand GSK3 or an antigenic fragment thereof, (xii) CSP or an antigenicfragment thereof and LRR9 or an antigenic fragment thereof, (xiii) CSPor an antigenic fragment thereof, MSP5 or an antigenic fragment thereof,and GSK3 or an antigenic fragment thereof, or (xiv) CSP or an antigenicfragment thereof, MSP5 or an antigenic fragment thereof, and LLR9 or anantigenic fragment thereof; (b) determining whether said human bodyfluid sample comprises an antibody that is specifically immunoreactiveto said at least one recombinant polypeptide, wherein positive specificimmunoreactivity indicates a state of protective immunity in saidsubject.

Certain embodiments are directed to a method to assess the response to amalaria vaccine in a human subject comprising: (a) testing for thepresence of an antibody binding to at least one a polypeptide comprisingDOC2 or an antigenic fragment thereof or DOC2 or an antigenic fragmentthereof in combination with one of the following: (i) MSP5 or anantigenic fragment thereof, (ii) GSK3 or an antigenic fragment thereof,(iii) LRR9 or an antigenic fragment thereof, (iv) MSP5 or an antigenicfragment thereof and GSK3 or an antigenic fragment thereof, (v) MSP5 oran antigenic fragment thereof, LRR9 or an antigenic fragment thereof,(vi) UNKPROT or an antigenic fragment thereof and LRR9 or an antigenicfragment thereof, (vii) GSK3 or an antigenic fragment thereof and LRR9or an antigenic fragment thereof, (viii) MSP5 or an antigenic fragmentthereof, GSK3 or an antigenic fragment thereof, and LRR9 or an antigenicfragment thereof, (ix) CSP or an antigenic fragment thereof, (x) CSP oran antigenic fragment thereof and MSP5 or an antigenic fragment thereof,(xi) CSP or an antigenic fragment thereof and GSK3 or an antigenicfragment thereof, (xii) CSP or an antigenic fragment thereof and LRR9 oran antigenic fragment thereof, (xiii) CSP or an antigenic fragmentthereof, MSP5 or an antigenic fragment thereof, and GSK3 or an antigenicfragment thereof, or (xiv) CSP or an antigenic fragment thereof, MSP5 oran antigenic fragment thereof, and LLR9 or an antigenic fragment thereofin a body fluid sample taken from the subject after a first dose of amalaria vaccine is administered to the subject; and (b) determining thatthe subject has protective immunity against P. falciparum-inducedmalaria if the presence of antibody binding to the at least onerecombinant polypeptide from step (a) is detected.

Methods of Immunizing or Conferring Protective Immunity

Certain embodiments are directed to a method for immunizing orconferring protective immunity against Plasmodium-induced malaria,particularly Pf-induced malaria, to a subject comprising: (a)determining if an antibody that specifically immunoreacts with a Pfimmunologic determinant comprising (i) a polypeptide having a sequencethat is at least 85%, 90%, 95%, 99%, or 100% identical to an amino acidsequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or antigenicfragments thereof, or encoded by a polynucleotide selected from thegroup of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, an antigenic fragmentthereof, or any combination thereof is in a body fluid sample from saidsubject; and (b) administering a P. falciparum vaccine to the subject ifthe body fluid sample lacks the antibody that specifically immunoreactswith the Pf immunologic determinant of (a).

Another embodiment is directed to a method for immunizing or conferringprotective immunity against Plasmodium-induced malaria, particularlyPf-induced malaria, to a subject, wherein the method comprises: (a)determining if an antibody in a body fluid sample from said subjectspecifically immunoreacts with a Pf immunologic determinant comprising apolypeptide having a sequence that is at least 80%, 85%, 90%, 95%, 99%,or 100% identical to an amino acid sequence of SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, or SEQ ID NO:18, or antigenic fragments thereof, or encoded by apolynucleotide selected from the group of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ IDNO:8, an antigenic fragment thereof, or any combination thereof; (b)administering a P. falciparum vaccine to the subject having a sampledetermined to lack immunoreactivity in (a).

Another embodiment is directed to a method of immunizing or conferringprotective immunity against Plasmodium-induced malaria, particularlyPf-induced malaria, to a subject lacking an antibody that specificallyimmunoreacts with a Pf immunologic determinant, comprising administeringa P. falciparum vaccine to the subject, wherein the Pf immunologicdeterminant comprises a polypeptide comprising a sequence that is atleast 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence ofSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or antigenic fragmentsthereof, or encoded by a polynucleotide having a nucleic acid sequencechosen from the group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, anantigenic fragment thereof, or any combination thereof.

Another embodiment is directed to a method for treating a subjectlacking protective immunity against Plasmodium-induced malaria,particularly Pf-induced malaria, after being inoculated with a P.falciparum vaccine comprising: (a) inoculating a subject with a P.falciparum vaccine; (b) determining if an antibody in a body fluidsample from the subject specifically immunoreacts with a Pf immunologicdeterminant comprising a polypeptide comprising a sequence that is atleast 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence ofSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or antigenic fragmentsthereof, or encoded by a polynucleotide selected from the group of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, or SEQ ID NO:8, an antigenic fragment thereof, or anycombination thereof and (c) administering a dose of the P. falciparumvaccine to the subject if the sample lacks an antibody that specificallyimmunoreacts with the Pf immunologic determinant of (b).

In some embodiments, the Pf immunologic determinant comprises DOC2, or acombination of DOC2/MSP5, DOC2/GSK3, DOC2/LRR9, DOC2/MSP5/GSK3,DOC2/MSP5/LRR9, DOC2/UNKPROT/LRR9, DOC2/GSK3/LRR9, DOC2/MSP5/GSK3/LRR9,CSP/DOC2, CSP/DOC2/MSP5, CSP/DOC2/GSK3, CSP/DOC2/LRR9,CSP/DOC2/MSP5/GSK3, or CSP/DOC2/MSP5/LLR9, or antigenic fragmentsthereof.

In some embodiments, the sensitivity of the Pf immunological determinantis at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In some embodiments, the specificity of the Pf immunological determinantis at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

Certain embodiments are directed to a method for immunizing a subjectagainst Plasmodium-induced malaria, particularly Pf-induced malaria,comprising: (a) determining if a body fluid sample from a subjectcomprises an antibody that specifically immunoreacts with at least onerecombinant polypeptide having a sequence that is at least 85%, 90%,95%, 99%, or 100% identical to an amino acid sequence of SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, or SEQ ID NO:18, or antigenic fragments thereof, orencoded by a polynucleotide chosen from SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ IDNO:8, or an antigenic fragment thereof; and (b) administering a P.falciparum vaccine to the subject if the sample lacks the antibody thatspecifically immunoreacts with the at least one polypeptide of (a).

Certain embodiments are directed to a method for immunizing a subjectlacking an antibody that specifically immunoreacts with at least one Pfimmunologic determinants, comprising administering a P. falciparumvaccine to the subject, wherein the at least one Pf immunologicdeterminants is a recombinant polypeptide comprising a sequence that isat least 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequenceof SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or antigenic fragmentsthereof, or encoded by a polynucleotide having a nucleic acid sequencechosen from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or an antigenic fragmentthereof.

Certain embodiments are directed to a method for immunizing a subjectagainst Plasmodium-induced malaria, particularly Pf-induced malaria,comprising: (a) determining if a body fluid sample from a subjectcomprises an antibody that specifically immunoreacts with a polypeptidecomprising DOC2 or an antigenic fragment thereof or DOC2 or an antigenicfragment thereof in combination with one of the following: (i) MSP5 oran antigenic fragment thereof, (ii) GSK3 or an antigenic fragmentthereof, (iii) LRR9 or an antigenic fragment thereof, (iv) MSP5 or anantigenic fragment thereof and GSK3 or an antigenic fragment thereof,(v) MSP5 or an antigenic fragment thereof, LRR9 or an antigenic fragmentthereof, (vi) UNKPROT or an antigenic fragment thereof and LRR9 or anantigenic fragment thereof, (vii) GSK3 or an antigenic fragment thereofand LRR9 or an antigenic fragment thereof, (viii) MSP5 or an antigenicfragment thereof, GSK3 or an antigenic fragment thereof, and LRR9 or anantigenic fragment thereof, (ix) CSP or an antigenic fragment thereof,(x) CSP or an antigenic fragment thereof and MSP5 or an antigenicfragment thereof, (xi) CSP or an antigenic fragment thereof and GSK3 oran antigenic fragment thereof, (xii) CSP or an antigenic fragmentthereof and LRR9 or an antigenic fragment thereof, (xiii) CSP or anantigenic fragment thereof, MSP5 or an antigenic fragment thereof, andGSK3 or an antigenic fragment thereof, or (xiv) CSP or an antigenicfragment thereof, MSP5 or an antigenic fragment thereof, and LLR9 or anantigenic fragment thereof; and (b) administering a P. falciparumvaccine to the subject if the sample lacks the antibody thatspecifically immunoreacts with the at least one polypeptide of (a).

Certain embodiments are directed to a method for immunizing a subjectlacking an antibody that specifically immunoreacts with at least one Pfimmunologic determinant, comprising administering a P. falciparumvaccine to the subject, wherein the at least one Pf immunologicdeterminant is DOC2 or an antigenic fragment thereof or DOC2 or anantigenic fragment thereof in combination with one of the following: (i)MSP5 or an antigenic fragment thereof, (ii) GSK3 or an antigenicfragment thereof, (iii) LRR9 or an antigenic fragment thereof, (iv) MSP5or an antigenic fragment thereof and GSK3 or an antigenic fragmentthereof, (v) MSP5 or an antigenic fragment thereof, LRR9 or an antigenicfragment thereof, (vi) UNKPROT or an antigenic fragment thereof and LRR9or an antigenic fragment thereof, (vii) GSK3 or an antigenic fragmentthereof and LRR9 or an antigenic fragment thereof, (viii) MSP5 or anantigenic fragment thereof, GSK3 or an antigenic fragment thereof, andLRR9 or an antigenic fragment thereof, (ix) CSP or an antigenic fragmentthereof, (x) CSP or an antigenic fragment thereof and MSP5 or anantigenic fragment thereof, (xi) CSP or an antigenic fragment thereofand GSK3 or an antigenic fragment thereof, (xii) CSP or an antigenicfragment thereof and LRR9 or an antigenic fragment thereof, (xiii) CSPor an antigenic fragment thereof, MSP5 or an antigenic fragment thereof,and GSK3 or an antigenic fragment thereof, or (xiv) CSP or an antigenicfragment thereof, MSP5 or an antigenic fragment thereof, and LLR9 or anantigenic fragment thereof.

Vaccine Compositions

Pharmaceutical compositions comprising polypeptides having the aminoacid sequences disclosed herein, and methods of using these compositionsas the immunogen in prophylactic vaccines to prevent malaria areprovided. These compositions and methods are useful to generate immuneresponses and provide partial, enhanced, or full protection in human andother mammalian subjects not previously exposed to a malaria-causingpathogen, or exposed, but not fully protected. These compositions andvaccines are similarly useful to reduce the chance of developing adisease-producing infection from parasites that causes malaria,including species of Plasmodium having a human host range, particularlyP. falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi, P.coatneyi, P. cynomolgi, P. simium and the like, and reduce the chance ofbecoming ill when one is infected, reduce the severity of the illness,such as fever, when one becomes infected, reduce the concentration ofparasites in the infected person, or reduce mortality rates from malariain populations exposed to malaria parasites. In many cases even partialprotection or delay in the time it takes an immunized individual ascompared to a non-immunized individual to become infected with theparasites or ill from infection is beneficial. Similarly, a vaccinetreatment strategy that results in any of these benefits in about 30% ofa population may have a significant impact on the health of a communityand of the individuals residing in the community.

In certain embodiments, the application is directed to a compositioncomprising one, two, three, four or more Pf immunologic determinantseach having an amino acid sequence that is at least 80%, 85%, 90%, 95%,99% or 100% identical to a polypeptide with an amino acid sequence ofSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, or SEQ IDNO:20, or antigenic fragments thereof, or encoded by a polynucleotidewith the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:10, or antigenic fragments thereof.

In some embodiments of this application, the composition comprises two,three, four or more Pf specific polypeptides each having an amino acidsequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to apolypeptide with an amino acid sequence of SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:20, or antigenicfragments thereof, or encoded by a polynucleotide with the nucleic acidsequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:9, or SEQ ID NO:10, or antigenic fragments thereof.

In some embodiments, the composition comprises DOC2, or a combination ofDOC2/MSP5, DOC2/GSK3, DOC2/LRR9, DOC2/MSP5/GSK3, DOC2/MSP5/LRR9,DOC2/UNKPROT/LRR9, DOC2/GSK3/LRR9, DOC2/MSP5/GSK3/LRR9, CSP/DOC2,CSP/DOC2/MSP5, CSP/DOC2/GSK3, CSP/DOC2/LRR9, CSP/DOC2/MSP5/GSK3, orCSP/DOC2/MSP5/LLR9, or antigenic fragments thereof.

In some embodiments the vaccine comprises one or more recombinant orsynthetic subunit vaccine components, including but not limited to arecombinant protein, or synthetic polypeptides comprising at least oneof SEQ ID NO:11-20 or an antigenic fragment thereof.

In some embodiments the vaccine comprises a polynucleotide (e.g., DNA orRNA) encoding at least one of SEQ ID NO:1-10 or an antigenic fragmentthereof. In some embodiments, the polynucleotide is functionallyincorporated in a vector, recombinant virus, recombinant bacteria, orrecombinant parasite.

It is known that there is cross reactivity among the orthologousimmunogens of different species of Plasmodium, particularly among thePlasmodium species of human host range. Therefore, a vaccine comprisingan immunogen derived from P. falciparum is expected to confer a degreeof protection to malarias whose etiologies are Plasmodia of other humanhost range species, particularly P. vivax, P. ovale, P. malariae, and P.knowlesi.

Provided are methods for generation of an immune response and preventionof malaria in a subject. The methods comprise administering to thesubject a vaccine, which has been prepared aseptically and comprisespolypeptides or DNA having one or more of the amino acid sequencesdisclosed herein in an amount effective to generate an immune responseor to prevent malaria.

The subject to which the vaccine is administered in accordance withthese methods may be any human or other mammal, susceptible to infectionwith a malaria parasite. For such methods, administration can be via thealimentary tract, such as oral, or administration can be parenteral,including, but not limited to mucosal, intranasal, epidermal, cutaneous,intramuscular, subcutaneous, intradermal, submucosal, intravenous andthe like. Moreover, the administration may be by continuous infusion orby single or multiple boluses as well as delivery mediated bymicroneedles.

The prevention and/or treatment of malaria may be readily ascertained bythe skilled practitioner by means of evaluation of clinical orpathological manifestations associated with malarial infection, forexample elevated temperature, headache, fatigue, coma, or percent oferythrocytes parasitized. Thus, according to the methods of the presentinvention, the subject shows improved or absent clinical signs, symptomsor pathological manifestations of malaria following administration of avaccine comprising purified live attenuated Plasmodium sporozoites.

Effective and optimal doses and dosage ranges for vaccines andimmunogens can be determined using methods known in the art. Guidance asto appropriate dosages to achieve an anti-malarial effect is providedfrom the exemplified assays disclosed herein. More specifically, resultsfrom the immunization pattern described herein and in cited referencescan be extrapolated by persons having skill in the requisite art toprovide a test vaccination schedule. Volunteer subjects are inoculatedwith varying doses at scheduled intervals and test blood samples areevaluated for levels of protection against malaria upon subsequentchallenge with infective parasites. Such results can be used to refinean optimized immunization dose and dosage regimen (schedule) foreffective immunization of mammalian, specifically human, subjects. It isanticipated that optimized doses and dosage regimens will vary generallywith the general body mass of the subject and infants and small childrenwill require proportionally less immunogen than adults. Furthermore,optimized doses and dosage regimens vary depending on the mode ofadministration, with intra dermal, subcutaneous and intramuscularadministration requiring more immunogen than intravenous administration.Methods of formulating pharmaceutical compositions and vaccines are wellknown to those of ordinary skill in the art (see, e.g., Remington, TheScience and Practice of Pharmacy 21st Edition, Hendrickson, ed. (USIP:2005)). Comprehended by the invention are vaccine compositionscomprising the polypeptides disclosed herein, and in certain embodimentsthese polypeptides are integrated into vectors along with appropriatediluent and buffer. Diluents, commonly Phosphate Buffered Saline (PBS),or Normal Saline (NS), are of various buffer content pH and ionicstrength. Such compositions may also include an excipient such as serumalbumin, particularly human serum albumin. Serum albumin may be purifiedfrom naturally occurring sources such as human blood, or be produced byrecombinant DNA or synthesis technologies. Such compositions may alsoinclude additives such as anti-oxidants e.g., ascorbic acid, sodiummetabisulfite, and/or preservatives or cryopreservatives. Incorporationof the material into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes mayalso be used. This may also include Plasmodium-related DNA vaccines or arecombinant virus, such as adenovirus, that express Plasmodium-relatedproteins, as a prime and purified, attenuated sporozoites vaccine as aboost, or vice versa. It may also include sequential or mixedimmunization with attenuated Plasmodium species sporozoites and someform of erythrocytic stage parasites, including, killed and liveattenuated. A vaccine complex comprising separate components may bereferred to as a vaccine regimen, a prime/boost regimen, componentvaccine, a component vaccine kit or a component vaccine package,comprising separate vaccine components. For example, a vaccine complexmay comprise as a component, a vaccine comprising purified, aseptic,live attenuated sporozoites. The complex may additionally comprise oneor more recombinant or synthetic subunit vaccine components, includingbut not limited to recombinant protein, synthetic polypeptide, DNAencoding these elements per se or functionally incorporated inrecombinant virus, recombinant bacteria, or recombinant parasite. Avaccine component may also include aseptic attenuated axenic sporozoitesthat are allowed to develop to the early liver stage extracellularly.Toll-like receptor agonists may be included in the vaccine formulationor conjugated directly to the polypeptides to control and direct theirimmunogenicity.

Kits

Certain embodiments are directed to a kit for identifying a humansubject lacking immunization or protective immunity against P.falciparum-induced malaria comprising, in one or more containers, (a) atleast one recombinant polypeptide having a sequence that is at least80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence ofSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20, or antigenic fragments thereof, or encoded by a polynucleotidecomprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ IDNO:10, or an antigenic fragment thereof, wherein the at least onerecombinant polypeptide of (a) are immobilized on one or more solidsupports; and (b) an immunologic determinants-antibody detectionreagent.

In some embodiments, the at least one recombinant polypeptide is DOC2,or a combination of DOC2/MSP5, DOC2/GSK3, DOC2/LRR9, DOC2/MSP5/GSK3,DOC2/MSP5/LRR9, DOC2/UNKPROT/LRR9, DOC2/GSK3/LRR9, DOC2/MSP5/GSK3/LRR9,CSP/DOC2, CSP/DOC2/MSP5, CSP/DOC2/GSK3, CSP/DOC2/LRR9,CSP/DOC2/MSP5/GSK3, or CSP/DOC2/MSP5/LLR9, or antigenic fragmentsthereof.

EXAMPLES Example 1

A clinical trial testing the immunogenicity and efficacy of aseptic,radiation-attenuated, purified, cryopreserved sporozoites used as theimmunogen in a vaccine formulation (Sanaria® PfSPZ Vaccine, provided bySanaria Inc.), and administered by intravenous injection, resulted in 13individuals that were protected against controlled human malariainfection (CHMI) and 19 that were not protected (Table 4). In the groupreceiving the highest dosage (675,000 total PfSPZ), 6 out of 6individuals were protected; and the group receiving the next lowerdosage (540,000 total PfSPZ) had 6 out of 9 protected (Seder et al.,Science 2013).

TABLE 4 Protection efficacy Total PfSPZ per dosage of Protection % DoseDoses PfSPZ # volunteers # Protected Protection % Combined 7500 (low) 430000 3 0 0% 0% 6 45000 3 0 0% 30000 4 120000 9 1 11% 9% (medium) 6180000 2 0 0% 135000 (high) 4 540000 9 6 67% 80% 5 675000 6 6 100%

The 5-dose (675000 SPZ) group in which all subjects were protecteddiffered from other groups, e.g., these subjects received the highestoverall dosage of SPZ and there was a 7-week interval between the fourthand fifth doses. The sera from both protect and unprotected individualswas used to identify immunogenic determinants.

Example 2 P. falciparum Proteome Microarray Chip Fabrication

P. falciparum (Pf) genes, representing 91% of the Pf proteome, wereselected and cloned into the Escherichia coli (E. coli) expressionvector pXT7. Included among these genes were those corresponding to SEQID NOs:1-10. Custom polymerase chain reaction (PCR) primers comprising20-bp gene-specific sequences with 33-bp adapter sequences were used toamplify target amplicons from Pf genomic DNA. The adapter sequences,which flank the target amplicons, are homologous to the adaptersequences at the ends of the linearized T7 expression vector pXT7 intowhich they were cloned. The homology allows the amplified PCR productsto be cloned into the expression vector by in vivo homologousrecombination in competent DH5a cells. The resulting clone mixtures werethen verified by PCR using sequence specific primers and subsequentlysequenced. For proteins microarray chip fabrication, Pf proteins wereexpressed in E. coli-based cell-free in vitro transcription andtranslation (IVTT) system (Rapid Translation System 100 High Yield [RTS100 HY] kits from 5 PRIME, Gaithersburg, Md.) according to themanufacturer's instructions. Expressed Pf proteins were then printedonto nitrocellulose-coated glass AVID slides (Grace Bio-Labs, Inc.,Bend, Oreg.) using an OmniGrid Accent microarray printer (DigiLab, Inc.,Marlborough, Mass.). Fabricated proteins microarray chips were QC usingmonoclonal anti-polyhistidine (clone His-1; Sigma-Aldrich, St. Louis,Mo.) and anti-hemagglutinin (clone 3F10; Roche, Indianapolis, Ind.)antibodies.

Proteome Microchip Design

Two types of Pf proteome microarrays were used: a down-selectedmicroarray of highly reactive Pf proteins (Pf1000), and a whole proteomemicroarray with 91% coverage of the Pf proteome (PfWPM). Each Pf1000chip contained 1,087 Pf peptide fragments representing proteins from 861unique Pf genes, 32 IgG positive control spots and 16 spotted IVTTreactions without Pf ORFs (IVTT controls). For each Pf1000 chip, 8replicates were printed per microarray slide on 8 nitrocellulose “pads”.PfWPM chips contained 7,455 Pf peptide fragments, representing proteinsfrom 4,805 unique genes, 302 IgG positive control spots and 192 IVTTcontrol spots. For each PfWPM chip, 3 replicates were printed permicroarray slide on 3 pads. IgG positive control spots were included asan assay control, while IVTT control spots were included as asample-level normalization factor.

Serum Probing Using P. falciparum Protein Microarray

Sera from human trial subjects was collected no sooner than 7 days afteradministration of the last dose of the vaccine regimen, preferablywithin 10-14 days after administration of the last dose. Plasma sampleswere diluted 1:100 in a 3 mg/mL E. coli lysate solution in proteinarraying buffer (Maine Manufacturing, Sanford, Me., USA) and incubatedat room temperature for 30 min. Chips were rehydrated in blocking bufferfor 30 min. Blocking buffer was removed, and chips were probed withpre-incubated serum samples using sealed, fitted slide chambers toensure no cross-contamination of sample between pads. Chips wereincubated overnight at 4° C. with agitation. Chips were washed fivetimes with TBS-0.05% Tween 20, followed by incubation withbiotin-conjugated goat anti-human IgG (Jackson ImmunoResearch, WestGrove, Pa., USA) diluted 1:200 in blocking buffer at room temperature.Chips were washed three times with TBS-0.05% Tween 20, followed byincubation with streptavidin-conjugated SureLight P-3 (ColumbiaBiosciences, Frederick, Md., USA) at room temperature protected fromlight. Chips were washed three times with TBS-0.05% Tween 20, threetimes with TBS, and once with water. Chips were air dried bycentrifugation at 1,000×g for 4 min and scanned on a ScanArray ExpressHT spectral scanner (Perkin-Elmer, Waltham, Mass., USA), and spot andbackground intensities were measured using an annotated grid file(.GAL). Data were exported in Microsoft Excel.

Protein Microarray Data Analysis

Raw spot and local background fluorescence intensities, spot annotationsand sample phenotypes were imported and merged in the R statisticalenvironment, where all subsequent procedures were performed(www.r-project.org). Foreground spot intensities were adjusted by localbackground by subtraction, and negative values were converted to 1.Next, all foreground values were transformed using the base 2 logarithm(Log 2). The dataset was normalized to remove systematic effects bysubtracting the median signal intensity of the IVTT controls for eachsample. Since the IVTT control spots carry the chip, sample andbatch-level systematic effects, but also antibody background activity tothe IVTT system, this procedure normalizes the data and provides arelative measure of the specific antibody binding to the non-specificantibody binding to the IVTT controls (a.k.a. background). With thenormalized data, a value of 0.0 means that the intensity is no differentthan the background, and a value of 1.0 indicates a doubling withrespect to background.

A seropositivity threshold was established as twice the IVTT background,or a normalized signal of 1.0, which was more conservative than two orthree standard deviations above the mean of the IVTT controls. Reactiveantigens were defined as those that had seropositive responses in atleast 10% of the study population, or 4 subjects, at eitherpre-immunization or pre-CHMI time points. Non-reactive antigens werefiltered before group comparisons were performed.

A high coverage, Pf Whole Proteome Microarray (PfWPM) was used to testantibodies against a total of 7,455 whole or partial Pf proteins,representing proteins from 4,805 unique genes, or approximately 91% ofthe Pf proteome. Among those antigens, 1,567 were identified asimmunoreactive in PfSPZ-immunized volunteers, according to ourreactivity criteria of 10% seroprevalence for a given antigen. Thereactivity profile of all subjects at pre-immunization andpost-immunization (pre-CHMI) time points are shown in FIG. 1A, anddifference between pre- and post-immunization, or “Deltas” in FIG. 1B.

A subset of antigens were identified with an “Immunogenic Profile” frompaired T-tests (FIG. 2), from the linear regression of antibody Deltameasurements (Table 5 and FIG. 3), and from linear mixed effectsregression (not shown due to overlap). The dose of sporozoites wassignificantly associated with antibody levels in 31 antigens afteradjusting P-values for multiple testing to avoid false discoveries, alsoknown as correction for the false discovery rate (Table 5 “BH P-value”and FIG. 3 gray triangles).

TABLE 5 Antigens associated with number of sporozoites administered fordeltas by linear regression Antigen Gene ID Description CoefficientP-value BH P-value* PF3D7_0304600 circumsporozoite (CS) protein (CSP)0.064 5.5E−7 0.0005 PF3D7_0312400 glycogen synthase kinase 3 (GSK3)0.037 3.3E−6 0.002 PF3D7_1468100 conserved Plasmodium protein, 0.014 3E−5 0.008 unknown function PF3D7_1243900 double C2-likedomain-containing 0.022 3.3E−5 0.008 protein PF3D7_1030200 conservedPlasmodium protein, 0.026  7E−5 0.013 unknown function PF3D7_0204200conserved Plasmodium protein, 0.008 0.0001 0.016 unknown functionPF3D7_0930300 merozoite surface protein 1 (MSP1) 0.010 0.0001 0.016PF3D7_1124000 endoplasmic reticulum 0.006 0.0002 0.021 oxidoreductin,putative PF3D7_0904900 Cu2 -transporting ATPase, putative 0.010 0.00020.021 (CUP) PF3D7_0906700 leucine-rich repeat protein (LRR9) 0.0270.0002 0.021 PF3D7_1332200 conserved Plasmodium protein, 0.007 0.00020.021 unknown function PF3D7_1011700 DNA repair protein RAD23, putative0.005 0.0003 0.024 PF3D7_0404600 conserved Plasmodium membrane 0.0070.0003 0.024 protein, unknown function PF3D7_0215700 DNA-directed RNApolymerase II 0.007 0.0005 0.031 second largest subunit, putativePF3D7_1438800 conserved Plasmodium protein, 0.023 0.0005 0.031 unknownfunction PF3D7_0929400 high molecular weight rhoptry 0.008 0.0005 0.031protein 2 (RhopH2) PF3D7_1409800 RNA binding protein Bruno, putative0.007 0.0006 0.032 (HoBo) PF3D7_0726100 Plasmodium exported protein,0.017 0.0006 0.033 unknown function PF3D7_1227500 cyclin (CYC2) 0.0070.0007 0.035 PF3D7_1417200 NOT family protein, putative 0.010 0.00080.035 PF3D7_1139500 AAA family ATPase, putative 0.010 0.0009 0.035PF3D7_0206900 merozoite surface protein 5 (MSP5), 0.010 0.0009 0.035truncated PF3D7_1427600 CorA-like Mg2 transporter protein, 0.012 0.00090.035 putative PF3D7_1110200 pre-mRNA-processing factor 6, 0.013 0.0010.036 putative (PRPF6) PF3D7_1438000 eukaryotic translation initiation0.008 0.001 0.036 factor eIF2A, putative PF3D7_1247500 serine/threonineprotein kinase, 0.009 0.001 0.036 putative PF3D7_1122800 calciumdependent protein kinase 6 0.007 0.001 0.036 (CDPK6) PF3D7_1327300conserved Plasmodium protein, 0.007 0.001 0.036 unknown functionPF3D7_1432300 conserved Plasmodium protein, 0.006 0.001 0.036 unknownfunction PF3D7_1469600 biotin carboxylase subunit of acetyl 0.007 0.0010.036 CoA carboxylase, putative (ACC1) PF3D7_0301700 Plasmodium exportedprotein, 0.005 0.001 0.036 unknown function *BH = Benjamin-Hochbergadjustment for the false discovery rate.

Example 3

Among the 1,567 immunoreactive antigens, a subset of proteins wasidentified as useful for identifying of protected subjects, or being“Associated with Protection.” In addition to CSP and MSP5, pre-CHMIantibodies against antigens with high AUC were glycogen synthase kinase3, or GSK3 (gene id: PF3D7_0312400; AUC: 0.94, BH P-value=0.004) and aconserved protein of unknown function (PF3D7_1030200; AUC: 0.88, BHP-value=0.036). These antigens and other top antigen are shown in FIG.4.

For antibody Deltas (the difference between pre-immunization andpre-CHMI), we identified a leucine-rich repeat protein, called LRR9(PF3D7_0906700; AUC: 0.90, BH P-value=0.003), and a protein described asdouble C2-like domain-containing protein, or DOC2 (PF3D7_1243900; AUC:0.96, BH P-value=0.0005) with high AUC for protection. These antigensand other top antigens are shown in Table 6 and FIG. 5. As with thedown-selected chip, the CSP was a top candidate with a pre-CHMI AUC of0.93 (BH P-value=0.004) and AUC of 0.93 (BH P-value=0.003) for antibodyDeltas.

TABLE 6 Top 10 antigens by Wilcoxon Rank-Sum test between protected andsusceptible volunteers, Deltas Antigen Gene ID Description AUC P-valueBH P-value* PF3D7_1243900 double C2-like domain-containing protein 0.965.6E−7 0.0005 (DOC2) PF3D7_1030200 conserved Plasmodium protein, unknown0.94 5.2E−6 0.002 function PF3D7_0304600 circumsporozoite (CS) protein(CSP) 0.93 9.1E−6 0.003 PF3D7_0906700 leucine-rich repeat protein (LRR9)0.90 4.0E−5 0.009 PF3D7_0312400 glycogen synthase kinase 3 (GSK3) 0.897.7E−5 0.014 PF3D7_0828100 conserved Plasmodium protein, unknown 0.840.0007 0.114 function PF3D7_1302500 conserved Plasmodium protein,unknown 0.84 0.0009 0.115 function PF3D7_0930300 merozoite surfaceprotein 1 (MSP1) 0.83 0.001 0.124 PF3D7_1438800 conserved Plasmodiumprotein, unknown 0.83 0.001 0.124 function PF3D7_1011700 DNA repairprotein RAD23, putative 0.83 0.001 0.131 *BH = Benjamin-Hochbergadjustment for the false discovery rate.

Example 4

Next, Combinatorial Analysis was performed selecting the highest rankingfeature sets by AUC for combinations of two to thirty antigens. Thegeneralizability of each set of features was tested using LOOCV. 960models were tested on the data from the 32 subjects for pre-CHMIantibody levels or antibody Deltas. For antibody Deltas, the bestcombinations came from 21-antigen models shown in Table 7 and FIG. 6B(number of antigens in model with the highest LOOCV AUC). Notably,numerous smaller antibody models gave similar AUC in the LOOCV,indicating that a subset of the 21 antigens is probably contributing themost to the AUC, and that there may be antigens in the model thatincrease the AUC through random noise, and that these antigens may befiltered, as they are not likely to be validated. FIG. 6C illustratesthe combinatorial models applied to the pre-immunization time point,which should not bear any antibody combinations that differentiateprotected and susceptible individuals. Indeed, the LOOCV AUCs for mostantigen combinations fluctuate around 0.5. In contrast, the AUCs formodels fit to the training sample set or the full data set quicklyapproach 1.0 with addition of more antigens.

TABLE 7 Deltas top antibody combination associated with protection in alinear model Protected Susceptible Antigen Gene ID DescriptionCoefficient Means Means PF3D7_0704700 conserved Plasmodium membrane 2.020.085 −0.084 protein, unknown function PF3D7_1015900 enolase (ENO) 1.960.192 −0.011 PF3D7_0631800 Rifin 1.79 0.303 −0.275 PF3D7_0604100transcription factor with AP2 1.69 0.086 −0.026 domain(s) PF3D7_1468100conserved Plasmodium protein, 1.26 0.57 −0.072 unknown functionPF3D7_1369200 conserved Plasmodium protein, 1.01 0.052 −0.173 unknownfunction PF3D7_0304600 circumsporozoite (CS) protein 0.835 5.03 1.89(CSP) PF3D7_0828100 conserved Plasmodium protein, 0.775 0.665 −0.012unknown function PF3D7_0910400 selenide water dikinase, putative 0.5230.013 −0.21 PF3D7_1110200 pre-mRNA-processing factor 6, 0.353 0.425−0.175 putative (PRPF6) PF3D7_0422500 pre-mRNA-splicing helicase BRR2,0.332 0.256 −0.098 putative (BRR2) PF3D7_0206900 merozoite surfaceprotein 5 0.251 0.526 0.029 (MSP5) PF3D7_0728600 zinc finger, C3HC4type, putative 0.199 1.57 0.152 PF3D7_1243900 double C2-likedomain-containing 0.134 1.07 −0.076 protein PF3D7_1417200 NOT familyprotein, putative 0.045 0.341 −0.143 PF3D7_0905300 dynein heavy chain,putative 0.031 0.15 −0.087 PF3D7_1412200 conserved Plasmodium protein,0.002 0.073 −0.167 unknown function PF3D7_0701600 Pfmc-2TM Maurer'scleft two −0.297 −0.051 0.141 transmembrane protein PF3D7_0704800conserved Plasmodium protein, −1.07 −0.177 0.072 unknown functionPF3D7_1219300 erythrocyte membrane protein 1, −1.24 −0.298 −0.092 PfEMP1PF3D7_1141600 dolichol phosphate mannose −3.95 −0.115 0.101 synthase(DPM1)

Example 5

Using an inclusive approach to antigen selection, antibodies against 52P. falciparum antigens were identified that showed significant vaccineimmunogenicity and dose responsiveness (“Immunogenicity Profile”, had asignificant association with distinguishing protection or susceptibilityto CHMI (“Association with Protection”) or were found as a top signatureof protection in combinatorial analysis (“Combinatorial Analysis”).These are shown in Table 8. Antibody levels for each listed antigen wereindividually inspected and graded on immunogenicity, protection and AUC.Ten antigens were further identified from the set of 52 that fulfilledour criteria for the best candidates for further development (Table 9).Of those 10 antigens, 6 had the highest performance. The LOOCV AUCs forthe combination of top 6 protein hits was 0.89 for pre-CHMI antibodylevels and 0.82 for antibody Deltas, while addition of the next 4targets resulted in LOOCV AUCs of 0.91 and 0.81, respectively (FIG.7A-B).

TABLE 8 Total of 52 antigens down-selected from 1,567 immunoreactiveantigens. Criteria for down-selecting antigens was demonstration of asignificant immunogenicity profile, significant association withprotection or a component of optimal antigen combinations incombinatorial analysis. Gene ID Description Criteria For Selection 1PF3D7_0204200 conserved Plasmodium protein, Immunogenicity Profileunknown function 2 PF3D7_0206900 merozoite surface protein 5 (MSP5)Immunogenicity Profile 3 PF3D7_0206900 merozoite surface protein 5Immunogenicity Profile; (MSP5), truncated Associated With Protection;Combinatorial Analysis 4 PF3D7_0207500 serine repeat antigen 6 (SERA6)Immunogenicity Profile 5 PF3D7_0215700 DNA-directed RNA polymerase IIImmunogenicity Profile second largest subunit, putative 6 PF3D7_0301700Plasmodium exported protein, Immunogenicity Profile unknown function 7PF3D7_0304600 circumsporozoite (CS) protein Immunogenicity Profile;(CSP) Associated With Protection; Combinatorial Analysis 8 PF3D7_0312400glycogen synthase kinase 3 (GSK3) Immunogenicity Profile; AssociatedWith Protection; Combinatorial Analysis 9 PF3D7_0404600 conservedPlasmodium membrane Immunogenicity Profile protein, unknown function 10PF3D7_0422500 pre-mRNA-splicing helicase BRR2, Combinatorial Analysisputative (BRR2) 11 PF3D7_0508900 conserved Plasmodium protein,Immunogenicity Profile unknown function 12 PF3D7_0604100 transcriptionfactor with AP2 Combinatorial Analysis domain(s) 13 PF3D7_0631800 RifinCombinatorial Analysis 16 PF3D7_0701600 Pfmc-2TM Maurer's cleft twoCombinatorial Analysis transmembrane protein 14 PF3D7_0704700 conservedPlasmodium membrane Combinatorial Analysis protein, unknown function 15PF3D7_0704800 conserved Plasmodium protein, Combinatorial Analysisunknown function 17 PF3D7_0726100 Plasmodium exported protein,Immunogenicity Profile unknown function 18 PF3D7_0728600 zinc finger,C3HC4 type, putative Combinatorial Analysis 19 PF3D7_0828100 conservedPlasmodium protein, Combinatorial Analysis unknown function 20PF3D7_0904900 Cu2 -transporting ATPase, putative Immunogenicity Profile(CUP) 21 PF3D7_0905300 dynein heavy chain, putative CombinatorialAnalysis 22 PF3D7_0906700 leucine-rich repeat protein (LRR9)Immunogenicity Profile; Associated With Protection 23 PF3D7_0910400selenide water dikinase, putative Combinatorial Analysis 24PF3D7_0918900 gamma-glutamylcysteine synthetase Immunogenicity Profile(gammaGCS) 25 PF3D7_0929400 high molecular weight rhoptry ImmunogenicityProfile protein 2 (RhopH2) 26 PF3D7_0930300 merozoite surface protein 1(MSP1) Immunogenicity Profile 27 PF3D7_1011700 DNA repair protein RAD23,Immunogenicity Profile putative 28 PF3D7_1015900 enolase (ENO)Combinatorial Analysis 29 PF3D7_1030200 conserved Plasmodium protein,Immunogenicity Profile; unknown function Associated With Protection 30PF3D7_1110200 pre-mRNA-processing factor 6, Immunogenicity Profile;putative (PRPF6) Combinatorial Analysis 31 PF3D7_1122800 calciumdependent protein kinase 6 Immunogenicity Profile (CDPK6) 32PF3D7_1124000 endoplasraic reticulum Immunogenicity Profileoxidoreductin, putative 33 PF3D7_1139500.1 AAA family ATPase, putativeImmunogenicity Profile 34 PF3D7_1141600 dolichol phosphate mannoseCombinatorial Analysis synthase (DPM1) 35 PF3D7_1219300 erythrocytemembrane protein 1, Combinatorial Analysis PfEMP1 36 PF3D7_1221000histone-lysine N-methyltransferase, Immunogenicity Profile H3 lysine-4specific (SET10) 37 PF3D7_1227500 cyclin (CYC2) Immunogenicity Profile38 PF3D7_1243900 double C2-like domain-containing ImmunogenicityProfile; protein Associated With Protection; Combinatorial Analysis 39PF3D7_1247500 serine/threonine protein kinase, Immunogenicity Profileputative 40 PF3D7_1327300 conserved Plasmodium protein, ImmunogenicityProfile unknown function 41 PF3D7_1332200 conserved Plasmodium protein,Immunogenicity Profile unknown function 42 PF3D7_1369200 conservedPlasmodium protein, Combinatorial Analysis unknown function 43PF3D7_1409800 RNA binding protein Bruno, Immunogenicity Profile putative(HoBo) 44 PF3D7_1412200 conserved Plasmodium protein, CombinatorialAnalysis unknown function 45 PF3D7_1417200 NOT family protein, putativeImmunogenicity Profile; Combinatorial Analysis 46 PF3D7_1427600CorA-like Mg2 transporter protein, Immunogenicity Profile putative 47PF3D7_1431100 conserved Plasmodium protein, Immunogenicity Profileunknown function 48 PF3D7_1432300 conserved Plasmodium protein,Immunogenicity Profile unknown function 49 PF3D7_1438000 eukaryotictranslation initiation Immunogenicity Profile factor eIF2A, putative 50PF3D7_1438800 conserved Plasmodium protein, Immunogenicity Profileunknown function 51 PF3D7_1468100 conserved Plasmodium protein,Immunogenicity Profile; unknown function Combinatorial Analysis 52PF3D7_1469600 biotin carboxylase subunit of acetyl ImmunogenicityProfile CoA carboxylase, putative (ACC1)

TABLE 9 List of ten antigens for identifying PfSPZ vaccine protection.Nucleic Acid Protein Antigen Gene ID Description Sequence SequencePF3D7_0304600 circumsporozoite (CS) SEQ ID NO: 9 SEQ ID NO: 19 protein(CSP) PF3D7_0206900 merozoite surface protein 5 SEQ ID NO: 10 SEQ ID NO:20 (MSP5) PF3D7_1030200 conserved Plasmodium SEQ ID NO: 1 SEQ ID NO: 11protein, unknown function PF3D7_0312400 glycogen synthase kinase 3 SEQID NO: 4 SEQ ID NO: 14 (GSK3) PF3D7_1243900 double C2-like domain- SEQID NO: 3 SEQ ID NO: 13 containing protein (DOC2) PF3D7_0906700leucine-rich repeat protein SEQ ID NO: 2 SEQ ID NO: 12 (LRR9)PF3D7_1438800 conserved Plasmodium SEQ ID NO: 5 SEQ ID NO: 15 protein,unknown function PF3D7_0828100 conserved Plasmodium SEQ ID NO: 6 SEQ IDNO: 16 protein, unknown function PF3D7_0728600 zinc finger, C3HC4 type,SEQ ID NO: 7 SEQ ID NO: 17 putative PF3D7_1468100 conserved PlasmodiumSEQ ID NO: 8 SEQ ID NO: 18 protein, unknown function

Example 6

An additional set of P. falciparum whole proteome microarray experimentswere run on a set of samples derived from an independent clinical trialof PfSPZ Vaccine, “Study of Controlled Human Malaria Infections toEvaluate Protection After Intravenous or Intramuscular Administration ofPfSPZ Vaccine in Malaria-Naive Adults” (ClinicalTrials.gov Identifier:NCT02015091). Serum samples assayed were from the trial arm receiving 3doses of 9×10⁵ PfSPZ Vaccine and given controlled human malariainfection (CHMI), for which 9 out of 14 volunteers were protected.

As a validation data set, only the 6 Pf antigens (PF3D7_1030200 (SEQ IDNO:11); LRR9 (SEQ ID NO:12); DOC2 (SEQ ID NO:13); GSK3 (SEQ ID NO:14);CSP (SEQ ID NO:19); and MSP5 (SEQ ID NO:20) were assessed forcorrelation with protection. These statistical methods used to determineassociation with sterile protection after CHMI: 1) classification ofsubjects as “protected” or “susceptible” using fitted generalized linearmodels of antibody measurements after third vaccination and before CHMIand antibody Deltas (baseline to post-third vaccination and baseline topre-CHMI) on the probability of being protected (logistic regression);2) Estimation of the area under the ROC curve (AUC) in theclassification of subjects as “protected” or “susceptible” as comparedto the true diagnosis of protected or susceptible according to the goldstandard of thick and thin blood smear by microscopy (as part of theCHMI protocol); 3) calculation of sensitivity and specificity of the 6Pf antigen combinatorial model.

Antibody levels measured after the third vaccination (“Peak”) were ableto classify subjects with an AUC of 1.0, while Peak antibody Deltas hadan AUC of 0.91. Antibody levels measured before CHMI (“Pre-CHMI”)classified subjects with an AUC of 1.0, while Pre-CHMI Deltas had an AUCof 0.89 (FIG. 8). The performance characteristics (sensitivity,specificity, false positives and false negatives) for the 6 Pf antigencombination are shown in Table 10 for 6-antigen combinations and inTables 11-12 for various combinations of the 6 Pf antigens.

TABLE 10 Performance characteristics of Peak antibody levels, Peakantibody Deltas (difference between pre-immunization andpost-immunization immunoreactivities), Pre-CHMI antibody levels andPre-CHMI antibody Deltas against combinations of 6 Pf antigens indiscriminating protection and susceptibility to CHMI. Predictive FalseFalse Antigens Value * Sensitivity Specificity Positives Negatives PeakAntibody Levels 9.47 100% 100% 0 0 Peak Antibody Deltas −2.04  89%  80%1 1 Pre-CHMI Antibody Levels 163 100% 100% 0 0 Pre-CHMI Antibody Deltas0.76  89% 100% 0 1 * Predictive Value: The predictive value displayed isthe optimal logistic regression prediction (“predictive value”) formaximizing sensitivity and specificity performance characteristics indetermining protection against P. falciparum malaria.

TABLE 11 Performance characteristics of 6 Pf antigens using Peakantibody levels, Peak antibody Deltas (difference betweenpre-immunization and post-immunization immunoreactivities), Pre-CHMIantibody levels and Pre-CHMI antibody Deltas against combinations of 6Pf antigens in discriminating protection and susceptibility to CHMI.Peak Levels Peak Deltas Pre-CHMI Pre-CHMI Sensi- Sensi- Levels DeltasAntigens tivity Specificity tivity Specificity Sensitivity SpecificitySensitivity Specificity CSP 0.67 0.40 0.89 0.40 0.56 0.60 0.89 0.60 MSP50.56 0.60 0.67 0.80 0.44 0.60 0.67 0.60 PF3D7_1030200 0.78 0.60 0.780.60 0.56 0.60 0.44 0.60 GSK3 0.78 0.60 0.78 0.60 0.67 0.60 0.44 0.60DOC2 0.89 0.60 0.89 0.60 0.89 0.60 0.89 0.60 LRR9 0.78 0.80 0.78 0.800.89 0.60 0.78 0.80 CSP, MSP5 0.44 0.60 0.78 0.80 0.89 0.60 0.78 0.60CSP, PF3D7_1030200 0.56 0.60 0.44 0.60 0.67 0.60 0.78 0.60 CSP, GSK30.67 0.80 0.78 0.60 0.78 0.60 0.89 0.60 CSP, DOC2 0.78 0.80 0.67 0.600.89 0.60 0.78 0.60 CSP, LRR9 0.78 0.80 0.78 0.80 0.67 0.80 0.78 0.80MSP5, PF3D7_1030200 0.89 0.60 0.78 0.80 0.67 0.60 0.56 0.60 MSP5, GSK30.89 0.60 0.78 0.80 0.89 0.60 0.44 0.60 MSP5, DOC2 0.67 0.80 0.67 0.800.78 0.80 0.78 0.60 MSP5, LRR9 0.78 0.80 0.78 0.80 0.89 0.60 0.78 0.80PF3D7_1030200, GSK3 0.78 0.60 0.78 0.60 0.44 0.60 0.44 0.60PF3D7_1030200, DOC2 0.89 0.80 0.67 0.80 0.67 0.80 0.78 0.60PF3D7_1030200, LRR9 0.78 0.80 0.89 0.80 0.67 0.80 0.78 0.80 GSK3, DOC20.89 0.80 0.67 0.80 1.00 0.60 0.78 0.60 GSK3, LRR9 0.78 0.60 0.67 0.800.78 0.80 0.78 0.80 DOC2, LRR9 0.89 0.80 0.78 0.80 0.67 0.80 0.78 0.80CSP, MSP5, PF3D7_1030200 0.44 0.60 0.78 0.80 0.89 0.60 0.89 0.60 CSP,MSP5, GSK3 0.78 0.60 0.78 0.80 0.78 0.80 0.78 0.60 CSP, MSP5, DOC2 0.780.80 0.67 0.80 0.78 0.80 0.78 0.80 CSP, MSP5, LRR9 0.78 0.80 0.67 0.800.78 0.80 0.89 1.00 CSP, PF3D7_1030200, GSK3 0.67 0.80 0.67 0.60 0.670.60 0.44 0.60 CSP, PF3D7_1030200, DOC2 0.89 0.80 0.78 0.80 1.00 0.600.67 0.80 CSP, PF3D7_1030200, LRR9 0.89 1.00 0.89 0.80 0.89 1.00 0.780.80 CSP, GSK3, DOC2 0.89 0.80 0.78 0.80 0.78 0.60 0.78 0.60 CSP, GSK3,LRR9 0.78 0.80 0.67 0.80 0.67 0.80 0.78 0.80 CSP, DOC2, LRR9 0.89 0.800.78 0.80 0.89 0.80 0.78 0.80 MSP5, PF3D7_1030200, GSK3 1.00 0.60 0.780.80 0.89 0.60 0.56 0.60 MSP5, PF3D7_1030200, DOC2 0.89 0.80 0.78 0.800.78 0.80 0.78 0.60 MSP5, PF3D7_1030200, LRR9 0.67 0.80 0.89 0.80 0.890.80 0.78 0.80 MSP5, GSK3, DOC2 0.89 0.80 0.78 0.80 0.78 0.80 0.78 0.60MSP5, GSK3, LRR9 0.78 0.80 0.78 0.80 0.78 0.80 0.89 0.80 MSP5, DOC2,LRR9 0.89 0.80 0.78 0.80 0.89 1.00 0.89 0.80 PF3D7_1030200, GSK3, DOC20.89 0.80 0.67 0.80 0.89 0.60 0.78 0.60 PF3D7_1030200, GSK3, LRR9 0.780.80 0.89 0.80 0.89 0.80 0.78 0.80 PF3D7_1030200, DOC2, LRR9 0.78 0.800.89 0.80 0.89 0.80 0.89 0.80 GSK3, DOC2, LRR9 0.89 0.80 0.78 0.80 0.780.80 0.78 0.80 CSP, MSP5, PF3D7_1030200, GSK3 0.67 0.60 0.78 0.80 0.670.80 0.44 0.60 CSP, MSP5, PF3D7_1030200, DOC2 0.89 0.80 0.78 0.80 0.780.80 0.78 0.80 CSP, MSP5, PF3D7_1030200, LRR9 0.89 1.00 0.78 0.80 1.001.00 0.89 1.00 CSP, MSP5, GSK3, DOC2 0.89 0.80 0.78 0.80 1.00 0.60 0.780.80 CSP, MSP5, GSK3, LRR9 0.78 0.80 0.89 0.80 0.89 1.00 0.89 1.00 CSP,MSP5, DOC2, LRR9 0.78 0.80 0.67 0.80 0.89 1.00 0.89 1.00 CSP,PF3D7_1030200, GSK3, DOC2 0.89 0.80 0.78 0.80 0.67 0.60 0.89 0.60 CSP,PF3D7_1030200, GSK3, LRR9 1.00 1.00 0.89 0.80 1.00 1.00 0.78 0.80 CSP,PF3D7_1030200, DOC2, LRR9 0.89 1.00 0.89 1.00 0.89 1.00 0.89 0.80 CSP,GSK3, DOC2, LRR9 1.00 0.80 0.89 0.80 0.89 0.80 0.78 0.80 MSP5,PF3D7_1030200, GSK3, DOC2 0.89 0.80 0.78 0.80 0.78 0.80 0.78 0.60 MSP5,PF3D7_1030200, GSK3, LRR9 0.78 0.80 0.89 0.80 0.89 0.80 0.89 0.80 MSP5,PF3D7_1030200, DOC2, LRR9 0.78 0.80 0.89 0.80 1.00 1.00 0.89 0.80 MSP5,GSK3, DOC2, LRR9 0.78 0.80 0.89 0.80 1.00 1.00 0.89 0.80 PF3D7_1030200,GSK3, DOC2, LRR9 0.78 0.80 0.89 0.80 0.89 1.00 0.89 0.80 CSP, MSP5,PF3D7_1030200, GSK3, DOC2 0.89 0.80 0.78 0.80 1.00 0.60 0.67 0.80 CSP,MSP5, PF3D7_1030200, GSK3, LRR9 1.00 1.00 0.89 0.80 1.00 1.00 0.89 1.00CSP, MSP5, PF3D7_1030200, DOC2, LRR9 1.00 1.00 0.78 0.80 1.00 1.00 0.891.00 CSP, MSP5, GSK3, DOC2, LRR9 0.89 0.80 0.89 0.80 1.00 1.00 0.89 1.00CSP, PF3D7_1030200, GSK3, DOC2, LRR9 1.00 1.00 0.89 1.00 1.00 1.00 0.890.80 MSP5, PF3D7_1030200, GSK3, DOC2, 1.00 1.00 0.89 0.80 1.00 1.00 0.890.80 LRR9 CSP, MSP5, PF3D7_1030200, GSK3, 1.00 1.00 0.89 0.80 1.00 1.000.89 1.00 DOC2, LRR9

TABLE 12 Performance characteristics of 6 Pf antigens using Peakantibody Deltas (difference between pre-immunization andpost-immunization immunoreactivities) and Pre-CHMI antibody Deltas(difference between pre-immunization and pre-CHMI immunoreactivities)against combinations of 6 Pf antigens in discriminating protection andsusceptibility to CHMI. Peak Deltas Pre-CHMI Deltas Antigens SensitivitySpecificity Sensitivity Specificity CSP 0.89 0.40 0.89 0.60 MSP5 0.670.80 0.67 0.60 PF3D7_1030200 0.78 0.60 0.44 0.60 GSK3 0.78 0.60 0.440.60 DOC2 0.89 0.60 0.89 0.60 LRR9 0.78 0.80 0.78 0.80 CSP, MSP5 0.780.80 0.78 0.60 CSP, PF3D7_1030200 0.44 0.60 0.78 0.60 CSP, GSK3 0.780.60 0.89 0.60 CSP, DOC2 0.67 0.60 0.78 0.60 CSP, LRR9 0.78 0.80 0.780.80 MSP5, PF3D7_1030200 0.78 0.80 0.56 0.60 MSP5, GSK3 0.78 0.80 0.440.60 MSP5, DOC2 0.67 0.80 0.78 0.60 MSP5, LRR9 0.78 0.80 0.78 0.80PF3D7_1030200, GSK3 0.78 0.60 0.44 0.60 PF3D7_1030200, DOC2 0.67 0.800.78 0.60 PF3D7_1030200, LRR9 0.89 0.80 0.78 0.80 GSK3, DOC2 0.67 0.800.78 0.60 GSK3, LRR9 0.67 0.80 0.78 0.80 DOC2, LRR9 0.78 0.80 0.78 0.80CSP, MSP5, PF3D7_1030200 0.78 0.80 0.89 0.60 CSP, MSP5, GSK3 0.78 0.800.78 0.60 CSP, MSP5, DOC2 0.67 0.80 0.78 0.80 CSP, MSP5, LRR9 0.67 0.800.89 1.00 CSP, PF3D7_1030200, GSK3 0.67 0.60 0.44 0.60 CSP,PF3D7_1030200, DOC2 0.78 0.80 0.67 0.80 CSP, PF3D7_1030200, LRR9 0.890.80 0.78 0.80 CSP, GSK3, DOC2 0.78 0.80 0.78 0.60 CSP, GSK3, LRR9 0.670.80 0.78 0.80 CSP, DOC2, LRR9 0.78 0.80 0.78 0.80 MSP5, PF3D7_1030200,GSK3 0.78 0.80 0.56 0.60 MSP5, PF3D7_1030200, DOC2 0.78 0.80 0.78 0.60MSP5, PF3D7_1030200, LRR9 0.89 0.80 0.78 0.80 MSP5, GSK3, DOC2 0.78 0.800.78 0.60 MSP5, GSK3, LRR9 0.78 0.80 0.89 0.80 MSP5, DOC2, LRR9 0.780.80 0.89 0.80 PF3D7_1030200, GSK3, DOC2 0.67 0.80 0.78 0.60PF3D7_1030200, GSK3, LRR9 0.89 0.80 0.78 0.80 PF3D7_1030200, DOC2, LRR90.89 0.80 0.89 0.80 GSK3, DOC2, LRR9 0.78 0.80 0.78 0.80 CSP, MSP5,PF3D7_1030200, GSK3 0.78 0.80 0.44 0.60 CSP, MSP5, PF3D7_1030200, DOC20.78 0.80 0.78 0.80 CSP, MSP5, PF3D7_1030200, LRR9 0.78 0.80 0.89 1.00CSP, MSP5, GSK3, DOC2 0.78 0.80 0.78 0.80 CSP, MSP5, GSK3, LRR9 0.890.80 0.89 1.00 CSP, MSP5, DOC2, LRR9 0.67 0.80 0.89 1.00 CSP,PF3D7_1030200, GSK3, DOC2 0.78 0.80 0.89 0.60 CSP, PF3D7_1030200, GSK3,LRR9 0.89 0.80 0.78 0.80 CSP, PF3D7_1030200, DOC2, LRR9 0.89 1.00 0.890.80 CSP, GSK3, DOC2, LRR9 0.89 0.80 0.78 0.80 MSP5, PF3D7_1030200,GSK3, DOC2 0.78 0.80 0.78 0.60 MSP5, PF3D7_1030200, GSK3, LRR9 0.89 0.800.89 0.80 MSP5, PF3D7_1030200, DOC2, LRR9 0.89 0.80 0.89 0.80 MSP5,GSK3, DOC2, LRR9 0.89 0.80 0.89 0.80 PF3D7_1030200, GSK3, DOC2, LRR90.89 0.80 0.89 0.80 CSP, MSP5, PF3D7_1030200, GSK3, DOC2 0.78 0.80 0.670.80 CSP, MSP5, PF3D7_1030200, GSK3, LRR9 0.89 0.80 0.89 1.00 CSP, MSP5,PF3D7_1030200, DOC2, LRR9 0.78 0.80 0.89 1.00 CSP, MSP5, GSK3, DOC2,LRR9 0.89 0.80 0.89 1.00 CSP, PF3D7_1030200, GSK3, DOC2, LRR9 0.89 1.000.89 0.80 MSP5, PF3D7_1030200, GSK3, DOC2, LRR9 0.89 0.80 0.89 0.80 CSP,MSP5, PF3D7_1030200, GSK3, DOC2, LRR9 0.89 0.80 0.89 1.00

In the foregoing, the present invention has been described withreference to suitable embodiments, but these embodiments are only forpurposes of understanding the invention and various alterations ormodifications are possible so long as the present invention does notdeviate from the claims that follow.

1. A method of detecting antibodies that indicate a state of protectiveimmunity against P. falciparum-induced malaria in a human subject,comprising: (a) contacting a human serum sample from said subject with afirst recombinant polypeptide comprising a sequence that is at least85%, identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, orSEQ ID NO:18, or an antigenic fragment thereof; and (b) detectingantibodies that are specifically immunoreactive to said firstrecombinant polypeptide, wherein said immunoreactivity is above a cutoffvalue in Table 1 for said first recombinant polypeptide.
 2. A method ofdetecting antibodies to assess the response to a malaria vaccine in ahuman subject comprising: (a) inoculating the human subject with a P.falciparum-specific vaccine; (b) contacting a serum sample from saidsubject with a first recombinant polypeptide comprising a sequence thatis at least 85% identical to the amino acid sequence of SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, or SEQ ID NO:18, or an antigenic fragment thereof, whereinsaid serum sample is taken from a subject after at least one dose of aP. falciparum vaccine is administered to the subject; and (c) detectingantibodies that are immunoreactive to the first recombinant polypeptideis above a cutoff value in Table 1 for said recombinant polypeptide. 3.A method for immunizing a subject in need of protection against P.falciparum-induced malaria comprising: (a) determining if a serum samplefrom a subject comprises an antibody that specifically immunoreacts withat least a first recombinant polypeptide comprising a sequence that isat least 85% identical to the amino acid sequence of SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, or SEQ ID NO:18, or an antigenic fragment thereof; and (b)administering a P. falciparum vaccine to the subject if the sample lacksthe antibody detected above a cut off value in Table 1 for the firstrecombinant polypeptide. 4-7. (canceled)
 8. The method of claim 1,further comprising contacting the human serum sample with a secondrecombinant polypeptide comprising an amino acid sequence that is atleast 85% identical to the sequence of SEQ ID NO:19 or SEQ ID NO: 20, oran antigenic fragment thereof; and detecting antibodies that arespecifically immunoreactive to said second recombinant polypeptide,wherein said immunoreactivity is above a cutoff value in Table 1 forsaid second recombinant polypeptide. 9-15. (canceled)
 16. The method ofclaim 1, wherein the first recombinant polypeptide comprises a sequencethat is at least 85% identical to the amino acid sequence of SEQ IDNO:11, and the method further comprises contacting the human serumsample with a second recombinant polypeptide comprising a sequence thatis at least 85% identical to the amino acid sequence of SEQ ID NO:12,SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:
 20. 17. Themethod of claim 1, wherein the first recombinant polypeptide comprises asequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO:12, and the method further comprises contacting the humanserum sample with a second recombinant polypeptide comprising a sequencethat is at least 85% identical to the amino acid sequence of SEQ IDNO:11, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:
 20. 18.The method of claim 1, wherein the first recombinant polypeptidecomprises a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO:13, and the method further comprises contactingthe human serum sample with a second recombinant polypeptide comprisinga sequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:20.
 19. The method of claim 1, wherein the first recombinant polypeptidecomprises a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO:14, and the method further comprises contactingthe human serum sample with a second recombinant polypeptide comprisinga sequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:19, or SEQ ID NO:20. 20-23. (canceled)
 24. The method of claim 1, wherein said antibodyimmunoreactivity is determined by enzyme-linked immunosorbent assay(ELISA).
 25. The method of claim 1, wherein the first recombinantpolypeptide is affixed to a solid phase. 26-30. (canceled)
 31. Themethod of claim 1, wherein the first recombinant polypeptide provides asensitivity of at least about 92% and a specificity of at least about95%.
 32. The method of claim 1, wherein the first recombinantpolypeptide provides a sensitivity of about 100% and a specificity of atleast about 89%.
 33. (canceled)
 34. The method of claim 1, wherein thecutoff is a predictive value in Table
 2. 35. (canceled)
 36. Acomposition comprising (a) an immunogen comprising a first Pf-specificpolypeptide comprising a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQID NO:14, or an antigenic fragment thereof; and (b) a carrier. 37-39.(canceled)
 40. The composition of claim 36 wherein the first Pf-specificpolypeptide comprises a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO:11, and the composition furthercomprises a second Pf-specific polypeptide comprising a sequence that isat least 85% identical to the amino acid sequence of SEQ ID NO: 12, SEQIN NO: 13, SEQ IN NO: 14, SEQ ID NO: 19, or SEQ ID NO:
 20. 41. Thecomposition of claim 36 wherein the first Pf-specific polypeptidecomprises a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO:12, and the composition further comprises a secondPf-specific polypeptide comprising a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 11, SEQ IN NO: 13,SEQ IN NO: 14, SEQ ID NO: 19, or SEQ ID NO:
 20. 42. The composition ofclaim 36 wherein the first Pf-specific polypeptide comprises a sequencethat is at least 85% identical to the amino acid sequence of SEQ IDNO:13, and the composition further comprises a second Pf-specificpolypeptide comprising a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 11, SEQ IN NO: 12, SEQ IN NO: 14, SEQID NO: 19, or SEQ ID NO:
 20. 43. The composition of claim 36 wherein thefirst Pf-specific polypeptide comprises a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO:14, and thecomposition further comprises a second Pf-specific polypeptidecomprising a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO: 11, SEQ IN NO: 12, SEQ IN NO: 13, SEQ ID NO: 19,or SEQ ID NO:
 20. 44-63. (canceled)
 64. The method of claim 1, whereinthe first recombinant polypeptide comprises a sequence that is at least85% identical to the amino acid sequence of SEQ ID NO: 13, and whereinthe method further comprises contacting the human serum sample with oneor more of the following sequences: (i) a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 20 or an antigenicfragment thereof, and a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof;(ii) a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO: 20 or an antigenic fragment thereof, and asequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 12 or an antigenic fragment thereof; (iii) a sequence that isat least 85% identical to the amino acid sequence of SEQ ID NO: 11 or anantigenic fragment thereof, and a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 12 or an antigenicfragment thereof; (iv) a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereofand a sequence that is at least 85% identical to the amino acid sequenceof SEQ ID NO: 12 or an antigenic fragment thereof; (v) a sequence thatis at least 85% identical to the amino acid sequence of SEQ ID NO: 20 oran antigenic fragment thereof, a sequence that is at least 85% identicalto the amino acid sequence of SEQ ID NO: 14 or an antigenic fragmentthereof, and a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO: 12 or an antigenic fragment thereof; (vi) asequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 19 or an antigenic fragment thereof, and a sequence that isat least 85% identical to the amino acid sequence of SEQ ID NO: 20 or anantigenic fragment thereof; (vii) a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 19 or an antigenicfragment thereof, and a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof;(viii) a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO: 19 or an antigenic fragment thereof, and asequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 12 or an antigenic fragment thereof; (ix) a sequence that isat least 85% identical to the amino acid sequence of SEQ ID NO: 19 anantigenic fragment thereof, a sequence that is at least 85% identical tothe amino acid sequence of SEQ ID NO: 20 or an antigenic fragmentthereof, and a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO: 14 or an antigenic fragment thereof; or (x) asequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 19 or an antigenic fragment thereof, a sequence that is atleast 85% identical to the amino acid sequence of SEQ ID NO: 20 or anantigenic fragment thereof, and a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 12 or an antigenicfragment thereof.
 65. The method of claim 64, wherein the cutoff valuefor the first recombinant polypeptide is selected from the cutoff valuesin Table
 1. 66. The method of claim 65, wherein the cutoff is apredictive value in Table
 2. 67-69. (canceled)
 70. A compositioncomprising (a) a first recombinant polypeptide comprising a sequencethat is at least 85% identical to the amino acid sequence of SEQ ID NO:13 or an antigenic fragment thereof, and (b) at least one of thefollowing combinations of recombinant polypeptides: (i) a recombinantpolypeptide comprising a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof,and a recombinant polypeptide comprising a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 14 GSK3 or anantigenic fragment thereof; (ii) a recombinant polypeptide comprising asequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 20 or an antigenic fragment thereof, and a recombinantpolypeptide comprising a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;(iii) a recombinant polypeptide comprising a sequence that is at least85% identical to the amino acid sequence of SEQ ID NO: 11 or anantigenic fragment thereof, and a recombinant polypeptide comprising asequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 12 or an antigenic fragment thereof; (iv) a recombinantpolypeptide comprising a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof,and a recombinant polypeptide comprising a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 12 or an antigenicfragment thereof; (v) a recombinant polypeptide comprising a sequencethat is at least 85% identical to the amino acid sequence of SEQ ID NO:20 or an antigenic fragment thereof, a recombinant polypeptidecomprising a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO: 14 or an antigenic fragment thereof, and arecombinant polypeptide comprising a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 12 or an antigenicfragment thereof; (vi) a recombinant polypeptide comprising a sequencethat is at least 85% identical to the amino acid sequence of SEQ ID NO:19 or an antigenic fragment thereof, and a recombinant polypeptidecomprising a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO: 20 or an antigenic fragment thereof; (vii) arecombinant polypeptide comprising a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 19 or an antigenicfragment thereof, and a recombinant polypeptide comprising a sequencethat is at least 85% identical to the amino acid sequence of SEQ ID NO:14 or an antigenic fragment thereof; (viii) a recombinant polypeptidecomprising a sequence that is at least 85% identical to the amino acidsequence of SEQ ID NO: 19 or an antigenic fragment thereof, and arecombinant polypeptide comprising a sequence that is at least 85%identical to the amino acid sequence of SEQ ID NO: 12 or an antigenicfragment thereof; (ix) a recombinant polypeptide comprising a sequencethat is at least 85% identical to the amino acid sequence of SEQ ID NO:19 an antigenic fragment thereof, a recombinant polypeptide comprising asequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 20 or an antigenic fragment thereof, and a recombinantpolypeptide comprising a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof;or (x) a recombinant polypeptide comprising a sequence that is at least85% identical to the amino acid sequence of SEQ ID NO: 19 or anantigenic fragment thereof, a recombinant polypeptide comprising asequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 20 or an antigenic fragment thereof, and a recombinantpolypeptide comprising a sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof.71. (canceled)